Epidemiological Significance of the Fox (Vulpes vulpes) in the Spread of Vector-Transmitted Zoonoses in the Area of Northern Croatia

Wild animals often serve as reservoirs for vector-borne zoonoses, which are on the rise worldwide but have not yet been sufficiently researched. Vector-borne zoonoses, such as those caused by <i>Anaplasma phagocytophilum</i>, <i>Borrelia burgdorferi</i> sensu lato, and <i>Dirofilaria immitis</i>, are a growing public health concern due to their increasing incidence and broad host range. The aim of this study was to determine the prevalence and risk factors for vector-borne bacterial (borreliosis, anaplasmosis, ehrlichiosis) and parasitic (dirofilariasis) pathogens and to detect some of these pathogens in the red fox (<i>Vulpes vulpes</i>) population in Croatia. A total of 179 blood samples from foxes from nine districts were analysed. The SNAP ^® 4Dx ^® Plus rapid test was used to detect circulating <i>D. immitis</i> antigen and antibodies against <i>B. burgdorferi</i>, <i>A. phagocytophilum</i>/<i>Anaplasma platys</i>, and <i>Ehrlichia canis</i>/<i>Ehrlichia ewingii</i>. Circulating <i>D. immitis</i> antigen was detected in 6.70% of the samples (95% CI: 3.20-10.19%), while antibodies against <i>A. phagocytophilum</i>/<i>A. platys</i> were found in 10.06% (95% CI: 5.8-14.25%). Only one sample was positive for <i>B. burgdorferi</i>, while no antibodies were detected for <i>E. canis</i>/<i>E. ewingii</i>. Spatial analysis revealed statistically significant differences in prevalence by geographical region (district) and age, while no significant correlations were found. In the standard PCR analysis, DNA of <i>D. immitis</i> was not detected in any of the eight positive and eight negative SNAP ^® 4Dx ^® Plus samples. <i>D. repens</i>, <i>A. reconditum</i>, or co-infections were also not detected by PCR. Of the nine samples that tested positive for <i>A. phagocytophilum</i>/<i>A. platys</i> antibodies, four were confirmed to be positive for <i>A. phagocytophilum</i> by nested and semi-nested PCR targeting the 16S rRNA and GroEL genes. Phylogenetic analysis revealed similarities with various European strains, including zoonotic strains. This study is the first molecular detection of <i>A. phagocytophilum</i> from blood samples of red foxes in Croatia. The results show that red foxes are not free from infections such as anaplasmosis and dirofilariasis, emphasising their possible role in the maintenance and transmission of these pathogens in certain regions of Croatia. These results underline the need for further research to better understand the epidemiological importance of red foxes in the spread of vector-borne diseases.