Update on tick-borne rickettsioses in mainland Portugal: emerging threats and potential vectors.

Background: Tick-borne rickettsioses (TBR) are emerging, neglected, zoonoses, caused by intracellular α-proteobacteria of the genus Rickettsia, that pose a growing public health concern. The aim of the present study was to evaluate rickettsial infections in questing ticks collected from four different ecological areas in mainland Portugal.

Methods: Over a two-year period, a total of 707 questing ticks were collected. Individual adult ticks and pooled nymphs were submitted to DNA extraction, followed by qPCR assays targeting the gltA rickettsial gene. Positive samples were then submitted to conventional PCR targeting the gltA and the ompA genes for phylogenetic analysis.

Results: In total, eight tick species were identified: Dermacentor marginatus, Haemaphysalis inermis, Haemaphysalis punctata, Hyalomma lusitanicum, Ixodes frontalis, Ixodes ricinus, Rhipicephalus pusillus, and Rhipicephalus sanguineus sensu lato. Additionally, rickettsial infection was associated with seven of these species, with I. frontalis being the exception. Notably, the prevalence of Rickettsia spp. was 26.35%, with phylogenetic validation confirming infections with R. helvetica, R. massiliae, R. monacensis, Candidatus R. rioja, and R. slovaca.

Conclusions: The present study highlights the necessity for ongoing surveillance to map and monitor both questing and feeding ticks, along with their vertebrate hosts. Effective control strategies are of utmost importance to mitigate the escalating threat of TBR. Additionally, the present study provides valuable epidemiological insights into TBR in Portugal, including the identification of R. slovaca infecting I. ricinus - an unconventional tick-pathogen relationship - and the first report of Candidatus R. rioja infecting D. marginatus in Portugal. In conclusion, this study contributes with valuable data regarding epidemiological results on ticks and TBR circulating in Portugal, emphasizing the importance of proactive measures to address this emerging public health challenge.