Leishmania infantum in red foxes (Vulpes vulpes): from clinical findings to cytokine expression

Background: The life cycle of Leishmania infantum is maintained mainly in dogs in anthropogenic environments and in many other wild animals in the sylvatic cycle. The ecological plasticity of some wild canids facilitates their role as hosts for Leishmania spp. in different endemic regions. Although red foxes (Vulpes vulpes) frequently test positive for L. infantum in Europe, little is known about their clinical presentation, immune response, or treatment outcomes. This study investigated the prevalence, clinical, and immunological features of L. infantum infection in foxes from southern Italy, complemented by an in vitro evaluation of cytokine responses in fox macrophages. Methods: Wild foxes from a wildlife rehabilitation center in southern Italy were molecularly and serologically screened for L. infantum. One sick fox underwent a complete diagnostic confirmation, treatment, and follow-up through hematological, biochemical, cytological, and molecular evaluations. In addition, peripheral blood mononuclear cells (PBMCs) isolated from a healthy fox were differentiated into macrophages and experimentally infected with L. infantum to assess early host-parasite interactions and cytokine gene expression profiles at two time points (4 h and 48 h). Results: Overall, 13 out of 54 foxes (24.1%) were molecularly positive for L. infantum, with a prevalence of 25% (5/20) in necropsied animals and 22% (9/41) in live animals. One individual tested seropositive for L. infantum and Ehrlichia sp. using the SNAP Leish 4Dx® test (2.4%, 1/41). The sick fox treated with meglumine antimoniate and allopurinol showed marked clinical and laboratory improvement. In the in vitro evaluation, the percentage of infected macrophages decreased from 32.8% at 4 h (2.25 parasites/cell) to 21.5% at 48 h (2 parasites/cell). Cytokine gene expression at 4 h and 48 h showed an increase in interleukin-6 (IL-6) (0.164-0.552) and IL-10 (0.828-4.245), stable IL-4 (0.505-0.708), a decrease in IL-12 (1.793-1.223) and IFN-γ (1.507-0.613), and consistently low TNF-α (0.377-0.411). Conclusions: The present study confirmed a high L. infantum prevalence of infection in red foxes from southern Italy. Serological findings herein and in the literature, together with in vitro cytokine gene expression, suggested that most foxes may remain subclinically infected. The high molecular positivity and the synanthropic nature of red foxes reinforce their role as reservoirs of L. infantum in endemic areas.