Prevalence of tick-borne pathogens in grey wolves (Canis lupus) in southwestern Europe

In Europe, studies on tick-borne pathogens (TBPs) in wolves are limited, primarily focusing on one population. This study aimed to assess the prevalence of TBPs in grey wolves (Canis lupus) from Spain (n=129), Italy (n=113), and Portugal (n=43) between 2001-2023. The presence of Anaplasma spp., Ehrlichia spp., Hepatozoon spp. and piroplasm DNA was analyzed in spleen samples, and risk factors for Hepatozoon canis prevalence were evaluated using a generalized linear model. Overall, 93.3% (266/285) wolves tested positive to at least one TBP, being H. canis the most prevalent (91.6%). Babesia canis (1.8%), Anaplasma phagocytophilum (1.4%), and Babesia capreoli (0.7%) were detected in small percentages and Ehrlichia spp. and Theileria spp. were not found. Coinfections involving H. canis + B. canis (1.8%) as well as A. phagocytophilum + B. capreoli (0.4%) were detected. Hepatozoon canis prevalence was significantly higher in Spain than in Portugal and it was higher in young compared to subadult individuals. The phylogenetic analysis showed the existence of well-defined clades of H. canis. The high prevalence of H. canis indicates that this pathogen is endemic in wolves from these three countries, whereas the importance of Anaplasma, Ehrlichia and piroplasm species seems to be negligible. Our results show a high nucleotide diversity among the H. canis sequences. In addition, our data reveals that wolves from the study countries are infected with TBPs, which could be of health concern. These findings highlight the importance of performing epidemiological studies in wildlife.