Characterization of Tick-Associated Microbiota in Spain Using a Nanopore-based Metabarcoding Approach: Insights into Potential Zoonotic Pathogens

Ticks, well-known as hematophagous vectors, play a crucial role in transmitting numerous zoonotic pathogens ranging from viruses and bacteria to protozoa. To prevent and understand the transition from tick-borne disease (TBD) endemicity to epidemicity, it is imperative to focus on epidemiological aspects to develop robust surveillance methods and strategies. In this study, we developed a Nanopore-based metabarcoding approach for bacteria detection in ticks collected from Catalunya and Valencia. A total of 186 adult ticks belonging to Rhipicephalus bursa, Dermacentor marginatus, Hyalomma lusitanicum, Hyalomma marginatum, and Ixoded ricinus species were studied. In this study, we used PCR amplification combined with Nanopore sequencing techniques and bioinformatics pipelines to characterize the tick- associated microbiota studies. Full-length 16S rRNA gene metabarcoding sequencing demonstrated 59 species and 336 genera generated belonging to 22 phyla, 33 classes, 111 orders, and 178 families. Within this examination, we identified some novel endosymbionts bacterial species that according to the best of our knowledge were not documented previously in Spain including Candidatus Coxiella mudrowiae, Francisella persica, Rickettsia africae, Rickettsia japonica, Rickettsia rhipicephali, and Unclassified Aeromonas. Candidatus Coxiella mudrowiae and Rickettsia africae species predominated in the Rhipicephalus bursa tick. The Francisella genus and Rickettsia rhipicephali species dominated in Hyalomma marginatum. While Rickettsia japonica is associated with all the examined tick samples except Rhipicephalus bursa. The alpha diversity results, as assessed by the Shannon index (p-value > 0.05), indicate that there are no statistically significant variations in the bacterial communities within ticks, whether we consider different geographic locations or the feeding status of the ticks.