Distribution of owners (<i>n</i> = 918) of seronegative dogs according to the socioeconomic and environmental conditions and understanding of the disease, Brazil 2008.

<p>*Brazilian minimum wages (Brazilian monthly minimum wage = U$262).</p

Municipality of Belo Horizonte, state of Minas Gerais, Brazil, subdivided into sanitary areas.

<p>Municipality of Belo Horizonte, state of Minas Gerais, Brazil, subdivided into sanitary areas.</p

Distribution of seronegative dogs (<i>n</i> = 1213) according to the characteristics of the animals, Brazil 2008.

<p>Distribution of seronegative dogs (<i>n</i> = 1213) according to the characteristics of the animals, Brazil 2008.</p

Hierarchical framework of risk factors for <i>Leishmania infantum</i> infection in dogs.

<p>Hierarchical framework of risk factors for <i>Leishmania infantum</i> infection in dogs.</p

Cellular profile of circulating lymphocytes and monocytes in dogs submitted to different vaccination protocols before and after challenge with <i>L. infantum</i> plus SGE.

<p>C (control; white square); Sap (saponin; white triagle); LB (killed <i>L. braziliensis</i> vaccine; black square), LBSap (killed <i>L. braziliensis</i> vaccine plus saponin; black triagnle): the <i>x</i>-axis displays the times at which the assays were conducted (Tbc: time before challenge with <i>L</i>. <i>infantum</i>; and 20, 90 274, 435, 541 and 885 days after challenge [dac] with <i>L</i>. <i>infantum</i>), and the <i>y</i>-axis represents the mean values of the (A) absolute counts of circulating lymphocytes and of (B) CD5⁺, (C) CD4⁺, (D) CD8⁺, (E) CD21⁺ cells and (F) CD14⁺ monocytes. Significant differences (<i>p</i><0.05) between the LBSap group and the control C, Sap and LB groups are indicated, respectively, by the letters a, b and c.</p

Anti-<i>Leishmania</i> reactivity in serum from dogs submitted to different vaccination protocols before and after intradermal challenge with <i>L. infantum</i> plus SGE: C (control; white square); Sap (saponin; white triangle); LB (killed <i>L. braziliensis</i> vaccine; black square); LBSap (killed <i>L. braziliensis</i> vaccine plus saponin; black triangle).

<p>IgG2/IgG1 ratio: C (control; white square) and LBSap (killed <i>L. braziliensis</i> vaccine plus saponin; black square). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049780#pone-0049780-g001" target="_blank">Figure 1A</a> represents anti-<i>L. infantum</i> total IgG, (B) anti-<i>L. infantum</i> IgG1, (C) anti-<i>L. infantum</i> IgG2 and (D) IgG2/IgG1 ratio: the <i>x</i>-axis displays the times at which the assays were conducted (Tbc: time before challenge with <i>L</i>. <i>infantum</i>; and 20, 90 274, 435, 541 and 885 days after challenge [dac] with <i>L</i>. <i>infantum</i>), and the <i>y</i>-axis represents the mean ELISA absorbance values determined at 492 nm in serum samples diluted 1∶80 for IgG total and subclasses. The cut-off is represented by the dotted line. Significant differences (<i>p</i><0.05) between the LBSap group and the control C, Sap and LB groups are indicated, respectively, by the letters a, b, and c.</p

Cell proliferation response of PBMCs after stimulation with (A) vaccine soluble antigen (VSA) and (B) soluble <i>L. infantum</i> antigen (SLcA).

<p>The middle and lower panels show the immunophenotypic profile of <i>in vitro</i> peripheral blood mononuclear cells following stimulation with (C) VSA and (D) SLcA determined at 90, 435 and 885 dac (days after challenge with <i>L. infantum</i> plus SGE) for vaccinated groups: C (control; white); Sap (saponin; white with weak hatched); LB (killed <i>L. braziliensis</i> vaccine; white with strong hatched); LBSap (killed <i>L. braziliensis</i> vaccine plus saponin; black). The results are expressed as index (stimulated cultures over non-stimulated cultures (SC/CC)) of the mean frequencies of CD5⁺, CD21⁺, CD4⁺ and CD8⁺ cells and MHC II expression in lymphocytes (reported as Mean Fluorescence Channel - MFC values). Significant differences (<i>p</i><0.05) between values measured at 90, 435 and 885 dac under the same group are indicated by connecting lines, and between the LBSap and the control C, Sap and LB groups are represented by the letters a, b and c, respectively.</p

Quantification of parasite burden in spleen samples at 885 dac (days after challenge with <i>L. infantum</i> plus SGE) for vaccinated groups: C (control; white); Sap (saponin; white with weak hatched); LB (killed <i>L. braziliensis</i> vaccine; white with strong hatched); LBSap (killed <i>L. braziliensis</i> vaccine plus saponin; black).

<p>(A) The quantification of amastigote forms of <i>Leishmania/</i>mg of spleen using real time PCR with specific primers for a single-copy gene of DNA polymerase of <i>Leishmania infantum</i>. (B) Parasitism reduction (%) in Sap, LB and LBSap in comparison with the control C group. Results were plotted representing median values for each group. Significant differences (<i>p</i><0.05) between the LBSap and the control C group are represented by the letter a.</p

Evaluation of cytokine mRNA levels in spleen samples at 885 dac (days after challenge with <i>L. infantum</i> plus SGE) for vaccinated groups: C (control; white); Sap (saponin; white with weak hatched); LB (killed <i>L. braziliensis</i> vaccine; white with strong hatched); LBSap (killed <i>L. braziliensis</i> vaccine plus saponin; black).

<p>The Log number of messenger RNA molecules for (A) IFN-γ, (B) TNF-α (C) IL-10 and (D) TGF- β1 and the cytokines ratio (E) IFN-γ/IL-10 and (F) IFN-γ/TGF-β1 are shown. Results were plotted representing median values for each group. Significant differences (<i>p</i><0.05) between the LBSap and the control C, Sap and LB groups are represented by the letters a, b and c, respectively.</p

Sequences of primers used for real-time PCR.

<p>Sequences of primers used for real-time PCR.</p