Tick-borne protozoa in dogs: Combined diagnostic approaches reveal unnoticed carriers of Hepatozoon canis and Theileria orientalis among owned dogs in Northen Italy

Apicomplexan haemoprotozoa, including piroplasms from the genera Babesia and Theileria, along with Hepatozoon spp., are tick-borne protozoans that affect domestic animals, wildlife, and humans. Employing a broaderspectrum 18S rDNA PCR assay for Apicomplexa haemoprotozoa, the present study investigated 155 owned dogs presented to the Veterinary Teaching Hospital of the University of Bologna and previously tested by routine diagnostic assays for Anaplasmataceae and Babesia spp. infection. The presence of three different species of Apicomplexan hemoprotozoa was observed. Specifically, Babesia canis was confirmed in two dogs, while the presence Hepatozoon canis (eight dogs), and Theileria orientalis (one dog) was also newly detected. Hepatozoon canis positive dogs mainly showed clinical history of comorbidities. Phylogenetic analysis highlighted genetic diversity among H. canis strains detected in this study, and from H. canis sequences from canid. Hosts. This study emphasizes the importance of using comprehensive diagnostic techniques to detect a wide range of hemoprotozoan infections, particularly in cases of vector-borne diseases with potential co-infections. This is the first report in Italy of T. orientalis in a canine host. Further researches are required to elucidate the role of dogs in the epidemiology and transmission of Theileria spp. and to explore the pathogenicity of Hepatozoon canis strains in the region.