First molecular detection of Ehrlichia canis in Croatia reveals its presence in rhipicephalus bursa ticks from ruminants

Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis, is considered endemic in Mediterranean Europe and is primarily associated with the brown dog tick, Rhipicephalus sanguineus sensu lato. In Croatia, previous molecular investigations in dogs failed to detect the pathogen, despite sporadic serological findings and the confirmed presence of competent tick vectors. The aim of this study was to investigate the presence of E. canis DNA in ticks collected in Croatia. A total of 583 ticks from five genera were collected from the environment, domestic animals, wildlife, and humans across approximately 130 locations. All ticks were screened by PCR targeting the 16S rRNA gene, and positive samples were further characterised by sequencing of the full-length 16S rRNA gene. Six ticks tested positive for Ehrlichia sp. DNA, and sequencing confirmed all isolates as E. canis. All positive ticks were identified as Rhipicephalus bursa and originated from coastal Mediterranean regions of Croatia. Positive specimens were collected from goats, sheep, and chamois (Rupicapra rupicapra). These findings represent the first molecular evidence of E. canis in Croatia and indicate its circulation in ruminant-associated tick populations within the Mediterranean part of the country. Further studies are needed to clarify transmission dynamics and the potential epidemiological relevance for domestic animals.