Seach for tick-borne agents and detection of a novel Ehrlichia sp. in ticks from wild rabbits in Northern Spain.

This study aimed to investigate tick-borne agents in ticks (Ixodida: Ixodidae) collected from wild rabbits (Oryctolagus cuniculus) hunted in northern Spain in 2022. A total of 120 ticks were collected from 11 rabbits (mean: 13.7 ticks/rabbit), and identified as 109 Rhipicephalus pusillus and 11 Haemaphysalis hispanica. DNA was extracted from 89 R. pusillus and 5 H. hispanica and tested by a battery of PCR assays targeting tick-borne bacteria (genera Rickettsia, Borrelia, Coxiella, family Ehrlichiaceae) and protozoa (Piroplasmida). All ticks were negative for these agents, except for two specimens (one H. hispanica and one R. pusillus) that yielded amplicons by the Ehrlichiaceae 16 S rRNA assay. These two tick specimens were further tested by PCR assays targeting fragments of the ehrlichial genes groEL and dsb, which were generated only from H. hispanica. The groEL haplotype showed the closest identity (95%) with over 30 Ehrlichia haplotypes, while the dsb haplotype showed closest identity (88%) with 'Candidiatus Ehrlichia pampeana'. Phylogenetic analyses inferred from the groEL, and dsb partial sequences indicated that the ehrlichial agent detected in a H. hispanica male- here designated as Ehrlichia sp. Hhisp1- is a novel agent, distinct from all ehrlichial agents with available sequences in GenBank. Our finding of Ehrlichia sp. Hhisp1 in H. hispanica points out the possibility that the wild rabbit could be a reservoir of this novel ehrlichial agent since there is no transovarial transmission of Ehrlichia spp., and H hispanica is highly specific to wild rabbits and their burrows.