Electrocardiographic evaluation of TcVac4-immunized dogs post challenge infection with <i>T</i>. <i>cruzi</i>.

<p>Dogs were vaccinated and challenged with <i>T</i>. <i>cruzi</i> as described in Materials and Methods. Cardiac hemodynamics were monitored by electrocardiography, and graded as 0–10, 10 being most severe. HAD, High axis deviation; LVC, Low Voltage Complex; ICP, Interventricular conduction Problems; RP, Repolarization Problems. Type II (Mobitz) AVB, 2^nd degree Atrio-Ventricular Block; ADR, Axis deviation to the right; Small QRS, reduced QRS wave; ND, Not determined (animals not included for the study of these parameters).</p><p>Electrocardiographic evaluation of TcVac4-immunized dogs post challenge infection with <i>T</i>. <i>cruzi</i>.</p

TcVac4-induced antibody response in dogs (± <i>T</i>. <i>cruzi</i>).

<p>Dogs were vaccinated with TcVac4 or TrIE only and infected with <i>T</i>. <i>cruzi</i>, as described in Materials and Methods. Shown are sera levels of <i>T</i>. <i>cruzi</i>-specific IgG <b>(A)</b>, IgG1 <b>(B)</b>, and IgG2 <b>(C)</b> antibody subtypes, determined by an ELISA. Dogs given pcDNA3.1/no infection and dogs given pcDNA3.1/<i>T</i>. <i>cruzi</i> were included as negative and positive controls, respectively. The serology time points are described as day -65 = basal response before immunization, day 0 representing antibody response after last immunization but before challenge infection, day 60 post challenge equivalent to acute infection phase, and day 365 post challenge equivalent to chronic disease phase. Each bar represents the absorbance mean value ± standard deviation. Within the same time point, statistical differences (p < 0.05) among groups are shown with different characters above the bars according to Tukey’s test.</p

Anatomopathological and histopathological abnormalities in dogs during the acute and chronic phases of <i>T</i>. <i>cruzi</i> infection and disease development (± TcVac4 vaccine).

<p>Anatomopathological and histopathological evaluations were conducted during acute (day 60 pi) and chronic (day 365 pi) phases of infection and disease development. Classification of abnormalities was conducted according to [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003625#pntd.0003625.ref025" target="_blank">25</a>] and [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003625#pntd.0003625.ref039" target="_blank">39</a>], and represented as-, None; +, slight; ++, moderate; and +++, severe; ND, Not determined (animals not included for the study of these parameters).</p><p>Anatomopathological and histopathological abnormalities in dogs during the acute and chronic phases of <i>T</i>. <i>cruzi</i> infection and disease development (± TcVac4 vaccine).</p

Frequency of parasite transmission from TcVac4-vaccinated dogs post challenge infection with <i>T</i>. <i>cruzi</i>.

<p>Dogs were vaccinated and challenged with <i>T</i>. <i>cruzi</i> as described in Materials and Methods. Triatomines (<i>Meccus longipennis</i>) were fed on dogs at day 36 pi and infection of insects was evaluated 60 days post-feeding.</p><p>Frequency of parasite transmission from TcVac4-vaccinated dogs post challenge infection with <i>T</i>. <i>cruzi</i>.</p

Blood parasitemia control in dogs immunized with TcVac4.

<p>Dogs were immunized with TcVac4 or TrIE, and infected with <i>T</i>. <i>cruzi</i> as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003625#pntd.0003625.g001" target="_blank">Fig 1</a>. Blood samples were evaluated for parasitemia by light microscopy at alternate days post-infection. Shown are data from day 16 to 46 after challenge infection, presented as mean values (n = 6/group). Different letters above lines show statistical differences (p< 0.05) among treatments within the same day of sampling according to Tukey’s test.</p

Macroscopic and microscopic alterations in the heart of <i>T</i>. <i>cruzi</i>-infected dogs (±TcVac4).

<p><b>(A)</b> Morphologic alterations of heart in acute infection phase (±TcVac4). Shown are representative heart pictures from dogs included in pcDNA3.1/no <i>Tc</i> (A), pcDNA3.1/<i>Tc</i> (B), TcVac4/<i>Tc</i> (C) and TrIE/<i>Tc</i> (D) groups, harvested at day 60 pi. Yellow arrows indicate right ventricular dilation. Red arrows indicate the pale/striated areas of fibrotic tissue. <b>(B)</b> Histological analysis of heart tissue-sections in acutely-infected dogs (±TcVac4). Heart tissue sections (5-μM) from left ventricle, septum, and right ventricle were obtained at 60 days pi, and stained with hematoxylin-eosin. Shown are representative micrographs of heart from dogs injected with pcDNA3.1/<i>Tc</i> (B, B1, B2), TcVac4/<i>Tc</i> (C, C1, C2), and TrIE/<i>Tc</i> (D, D1, D2). Micrographs from pcDNA3.1/no <i>Tc</i> (A, A1, A2) are shown as negative controls. Black arrows show amastigotes nests. White arrows show inflammatory infiltrate in the myocardium. <b>(C)</b> Morphologic alterations of heart in chronic disease phase (±TcVac4). Shown are representative heart pictures from dogs included in pcDNA3.1/no <i>Tc</i> (A), pcDNA3.1/<i>Tc</i> (B), TcVac4/<i>Tc</i> (C) and TrIE/<i>Tc</i> (D) groups, harvested at day 365 pi. Black arrows point out the right ventricular dilation. <b>(D)</b> Histological analysis of heart tissue-sections in chronically-infected dogs (±TcVac4). Heart tissue sections (5-μm) from left ventricle, septum, and right ventricle were obtained at 365 days pi, and stained with hematoxylin-eosin. Shown are representative micrographs of chronically-infected dogs injected with pcDNA3.1 only (B, B1, B2) or immunized with TcVac4 (C, C1, C2). Micrographs from pcDNA3.1/no <i>Tc</i>, i.e. normal/healthy controls (A, A1, A2) are shown for comparison. White arrows mark infiltrating inflammatory cells in myocardium.</p