, 2007 and Giunchetti et al., 2008a). LBSap vaccine is considered safe for administration, without induction of ulcerative lesions at the site of inoculation ( Giunchetti et al., 2007 and Vitoriano-Souza et al., 2008). Moreover, LBSap vaccinated dogs presented high IFN-γ and low IL-10 and TGF-β1 expression in the spleen, with significant reduction of parasite load in this organ ( Roatt Selleckchem Neratinib et al., 2012). Additionally, LBSap displayed a strong and sustained induction of humoral immune response, with increased levels of anti-Leishmania total IgG as well

as both IgG1 and IgG2, after experimental challenge ( Roatt et al., 2012). Considering the promising results of the LBSap vaccine, we aimed to further evaluate the immunogenicity biomarkers before and after experimental L. chagasi challenge. Thus, the profile of different cytokines (IL-4, IL-10, TGF-β, IL-12, IFN-γ, and tumor necrosis factor [TNF]-α) and

nitric oxide (NO) in supernatants of peripheral blood mononuclear cell (PBMC) cultures were evaluated before the first immunization (T0), 15 days after completion of the vaccine protocol (T3), and at time points 90 (T90) and 885 (T885) days after experimental L. chagasi challenge. The frequency of parasitism in the bone marrow was also evaluated until T885. Twenty male and female mongrel dogs that had been born and reared in the kennels of the Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil, were treated at 7 months with an anthelmintic and

vaccinated against rabies (Tecpar, Curitiba-PR, Brazil), Antidiabetic Compound Library cell assay canine distemper, type 2 adenovirus, Levetiracetam coronavirus, parainfluenza, parvovirus, and leptospira (Vanguard® HTLP 5/CV-L; Pfizer Animal Health, New York, NY, USA). The absence of specific anti-Leishmania antibodies was confirmed by indirect fluorescence immunoassay. Experimental dogs were divided into four experimental groups: (i) control (C) group (n = 5) received 1 ml of sterile 0.9% saline; (ii) LB group (n = 5) received 600 μg of L. braziliensis promastigote protein in 1 ml of sterile 0.9% saline; (iii) Sap group (n = 5) received 1 mg of saponin (Sigma Chemical Co., St. Louis, MO, USA) in 1 ml of sterile 0.9% saline; and (iv) LBSap group (n = 5) received 600 μg of L. braziliensis promastigote protein and 1 mg of saponin in 1 ml of sterile 0.9% saline. All animals received subcutaneous injections in the right flank at intervals of 4 weeks for a total of three injections. The challenge of experimental animals was performed after 100 days of vaccination protocol. In this sense, all dogs received intradermally 1.0 × 107 promastigotes of L. chagasi stationary phase of cultivation, in the inner side of the left ear, in addition to 5 acini of the salivary gland of L. longipalpis. This preliminary stage of the study was performed from 2005 to 2007. Promastigotes of L.