2.1. Animals

This study was submitted to the Committee for Animal Ethics of the University of Parma (approval number PROT. 06B-CE20 04/02/20), and the experiments were realized following the approved guidelines. Forty-five owned dogs with clinical manifestation and laboratory abnormalities (electropherogram and biochemical profile) related to leishmaniasis and never subjected to pharmacological treatments were enrolled from an endemic area in the south of Italy (Taranto). Blood was collected from the cefalic vein. Serum was separated by centrifugation within 15 min of collection. Plasma (lithium heparin) was collected and stored in cryovials (−80 °C) until redox status analyses were performed. All the dogs to be included in this study had to be negative for the in vitro serum tests for the detection of the Dirofilaria immitis antigen and for the antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis and Ehrlichia ewingii (SNAP 4D* Plus, Iddex Laboratories—Hoofddorp NL). Serum was assayed by ELISA (enzyme-linked immunosorbent assay) kit for the diagnosis of canine leishmaniasis (Leishmania Ab ELISA Biopronix Agrolabo S.p.A., Turin, Italy). Thereafter, all the samples with a positive result were subjected to the immunofluorescence antibody test (IFAT), and staged in four different groups in agreement with the Manual of Standards for Diagnostic Test and Vaccines proposed by the LeishVet group [22]. In particular, eight dogs were included in Stage I (antibody level 1:40–1:80) 9 dogs in Stage II (1:160–1:320), 11 dogs in Stage III (1:640–1:1280) and 17 dogs in Stage IV ( ≥1:2560). Twenty-four owned healthy dogs (Table 1), without clinical signs of leishmaniasis, all free from haemoparasitosis and with a negative outcome in the serological leishmania test, were enrolled as control from the nonendemic area (Parma). All the dogs enrolled in the present investigation had not been vaccinated against Leishmania.

The table reports the data (age, sex, weight) of the belonging to the control group.

F = female, M = male.