chagasi. To our knowledge, this is the first morphometrical approach of inflammation and the first report of occurrence of apoptosis in inflammatory cells in vivo involving natural infection with L. (L.) chagasi. A total of 16 positive and six negative-tested dogs previously examined for VL were used. Macroscopic skin lesions due to secondary infections in the pinna region were considered as criteria of exclusion. To confirm Autophagy inhibitor L. chagasi infection, blood samples were taken to detect anti-Leishmania antibodies by IFA and ELISA and needle aspiration of the popliteal lymph node and bone marrow was performed in each dog, to

direct visualization of the parasite and culture. Once confirmed the infection, they were euthanatized and submitted to necropsy for sample collection. Before fixation of the samples (spleen, liver, skin and lymph nodes), imprints of the cut surface on cleaned slides were taken to direct visualization of the parasite and confirm visceralization of the infection. Myelograms and imprints of popliteal lymph nodes, spleen, liver and skin were stained with Giemsa, for parasitological visualization ( Mikel, 1994).

Aspirates from spleen, liver, bone marrow and lymph node were also cultured for promastigotes in NNN-phase Schneider’s liquid medium. Polymerase Chain Reaction was performed to detect parasites only in pinna skin extracted DNA, using a target sequence of Leishmania donovani complex. Anti-Leishmania antibodies were selleck detected in all infected animals, the titers ranging from 1:40 through 1:640. All infected animals (symptomatic and asymptomatic) were positive in PCR and at least two of the three parasitological tests (Giemsa,

culture and immunohistochemistry) in different organs. Animals regarded as non-infected controls had negative results in all tests, including PCR. Efforts were made to avoid all unnecessary distress to the animals. Housing, anesthesia and all procedures concurred with the guidelines established by our local Institutional Animal Care and Use Committee that also reviewed and approved this work (CETEA, Universidade Federal de Minas Gerais, protocol n° 198/2007, approved on 03/27/08). Eight VL-positive Sitaxentan dogs (by serological and parasitological analysis) were used in this experiment, with the exception of the control group. Animals were divided into three groups: (a) Eight VL-positive animals with clinical signs of the disease; (b) eight positive animals, with no clinical signs; and (c) six VL-negative control animals. Standards used to group the animals followed the Pozio et al. (1981) classification. The animals were tranquilized with Acepromazine 1%, anesthetized with Sodium thiopental 2.5%. After this procedure, the animals were euthanatized with an overdose of sodium thiopental 7.5% (75 mg/kg) for further pos-mortem examination. Skin fragments from the pinna region were collected and routinely processed for histological analysis.