Parasite load of 73 skin fragments of a sample subjected to the immunohistochemical methods of streptavidin peroxidase, using hyperimmune serum from a dog naturally infected by <i>Leishmania infantum chagasi</i> (dilution 1∶100) and monoclonal antibody anti-<i>Leishmania</i> lipophosphoglycan (LPG) (Mo-anti LPG) (dilution 1∶100).

<p>Mann-Whitney p = 0.0001.</p

A–E: Fragment of skin of a patient with LCL, Caratinga, MG, Brazil.

<p>(A, B) Immunohistochemical labeling of amastigotes of <i>Leishmania</i> with an aliquot of a commercial monoclonal anti-<i>Leishmania</i> antibody and the streptavidin-biotin peroxidase method. (A) Low magnification showing a brown background evidenced by the cytoplasm of the epithelial layer cells (arrowheads). Bar = 32 µm. (B) Higher magnification showing intense non-specific staining visible as dark brown cytoplasmic staining of epithelial (arrowheads) and inflammatory mononuclear cells (macrophages) with intracellular amastigotes of <i>Leishmania</i> in the dermis (arrows) Bar = 16 µm. (C,D) Immunohistochemical labeling of amastigotes of <i>Leishmania</i> using dog hyperimmune serum as the primary antibody with the streptavidin-biotin peroxidase method (C) Low magnification showing light-blue stained background. Bar = 32 µm. (D) Higher magnification showing dark-brown-stained intracellular amastigotes of <i>Leishmania</i> within macrophages in the dermis (arrows) and light-blue-stained background. Bar = 16 µm; (A–D) Immunohistochemistry with the streptavidin peroxidase method counter-stained with Harris’s hematoxylin. (E) Observe immunolabeled amastigotes inside macrophages associated areas of tissue debris (arrow) Epithelium (Ep), Dermis (De).</p

A–D: Fragment of skin of a patient with LCL Caratinga, MG, Brazil.

<p>(A) Changes observed in the epidermis were intense acanthosis (AC) and papillomatosis (PL). Pearl corneas can also be seen (black arrow). Finger-like projections of epidermis into the dermis layer, papillomatosis (PL) Bar = 32 µm, (B) Higher magnification shows thickening of the spinous (acanthosis) layer due to proliferation of epidermal cells (arrowhead) leading to papillomatosis (PL). Bar = 16 µm, (C) Higher magnification showing the inflammatory infiltrate of mononuclear cells (plasma cells, macrophages and lymphocytes) in the dermis. Note Langhans-type giant cell formation, but without a typical granuloma formation (arrowhead). Bar = 16 µm. (D) Eosinophilic necrotic area in the dermis with fragmented collagen fibers resembling fibrinoid necrosis (arrowheads) Bar = 64 µm. Hematoxylin-eosin staining. Epithelium (Ep), Dermis (De), Papillomatosis (PL).</p

Cytokine profile of spleen T-lymphocytes from mice before (0) and at 7, 14, 28 and 42 days after infection with metacyclic (MT) or blood trypomastigotes (BT) of <i>Trypanosoma cruzi</i>.

<p>“Gray scale” diagrams were used to represent the cytokine pattern and the cytokine balance within T-cell subsets besides the overall cytokine balance with T-cells, highlighting the predominance of “low” cytokine-producers (white square), “high” TNF-α or IFN-γ producers (black square), “high” IL-10⁺ producers (light gray square) or “high” mixed cytokine-producers (dark gray square). Pie charts represent the percentage of animals displaying a given T-cells overall cytokine balance selectively amongst the “high” cytokine-producers. ND = Not detected.</p

Morphometric analysis and photomicrographs of the area of <i>T. cruzi</i> immunoreactions in the heart at 28 days after mice infection with metacyclic (MT; light gray bar) or blood trypomastigotes (BT; black bar) of <i>Trypanosoma cruzi</i>.

<p>Analysis of the area of <i>T. cruzi</i> amastigotes were identified by immunohistochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032912#s4" target="_blank">Material and Methods</a>. The results are expressed as parasited area ± standard error. Photomicrographs of the heart parasitism demonstrating isolated amastigotes in MT and typical amastigote nests in BT.</p

UPGMA.

<p>Phenogram constructed from UPGMA (Unweighted Pair Group Method using Arithmetic averages) resulting from the gene signature profile analysis obtained by LSSP-PCR of isolates obtained from hemocultures at 3 (Be-78is5-3mai, Be-78is21-3mai), 6 (Be-78is1-6mai; Be-78is2-6mai; Be-78is5-6mai; 78is15-6mai-Be) and 12 (Be-78is1-12mai; Be-78is15-12mai) months after infection of Swiss mice with 5000 blood trypomastigotes from Be-78 <i>Trypanosoma cruzi</i> strain in comparison with the Be-78 parental and Be-62 strains.</p

Host-Parasite Interactions in Chagas Disease: Genetically Unidentical Isolates of a Single <i>Trypanosoma cruzi</i> Strain Identified <i>In Vitro via</i> LSSP-PCR

<div><p>The present study aims at establishing whether the diversity in pathogenesis within a genetically diverse host population infected with a single polyclonal strain of <i>Trypanosoma cruzi</i> is due to selection of specific subpopulations within the strain. For this purpose we infected Swiss mice, a genetically diverse population, with the polyclonal strain of <i>Trypanosoma cruzi</i> Berenice-78 and characterized <i>via</i> LSSP-PCR the kinetoplast DNA of subpopulations isolated from blood samples collected from the animals at various times after inoculation (3, 6 and 12 months after inoculation). We examined the biological behavior of the isolates in acellular medium and <i>in vitro</i> profiles of infectivity in Vero cell medium. We compared the characteristics of the isolates with the inoculating strain and with another strain, Berenice 62, isolated from the same patient 16 years earlier. We found that one of the isolates had intermediate behavior in comparison with Berenice-78 and Berenice-62 and a significantly different genetic profile by LSSP-PCR in comparison with the inoculating strain. We hereby demonstrate that genetically distinct <i>Trypanosoma cruzi</i> isolates may be obtained upon experimental murine infection with a single polyclonal <i>Trypanosoma cruzi</i> strain.</p></div

Hemocultures.

<p>Be-78: Berenice-78; is: isolate; MAI/mai: months after infection.</p><p>Repartition of the positive hemocultures from blood samples collected from mice infected with Berenice-78 <i>T</i>. <i>cruzi</i> strains at three, six and twelve months after infection.</p

Infectivity and <i>in vitro</i> development illustration.

<p>Representative photomicrographs of <i>in vitro</i> assay infection in Vero cell cultures with Berenice-62 and Berenice-78 parental strains and subpopulations/isolates of Be-78 strain, obtained from hemocultures at 3, 6 or 12 months after infection of Swiss mice with 5000 blood trypomastigotes from Be-78 <i>Trypanosoma cruzi</i> strain. After 18 hours exposure to the parasite, the cells were washed to remove extracellular parasites and maintained in medium until collection, fixed and stained at 24, 48 or 72 hours after inoculation. Increasing the rate of infectivity (asterisks) 48h after incubation for Be-62 strain (a, b, c) strain and for Be 78is5-3mai isolate (g, h, i); Increased rate of intracellular multiplication (asterisks) in 72h time for Be-78 parental strain (d, e, f); Infectivity and intracellular development profiles upper (asterisks) for isolate Be-78is2-6mai (j, k. G), and lower for Be-78is14-12mai (m, n, o). Fast Panotic. Bar = 25μm.</p

Infectivity and in vitro development.

<p><i>In vitro</i> infection assay in Vero cell cultures using Berenice-62 (grey square), Berenice-78 (parental, black square) strains and subpopulations/isolates of Be-78 strain, obtained from hemocultures at 3, 6 or 12 months after infection of Swiss mice with 5000 blood trypomastigotes from Be-78 <i>Trypanosoma cruzi</i> strain. After 18 exposure hours to the parasite, the cells were washed to remove extracellular parasites and maintained in medium until collection, fixation and staining 24 (solid bar), 48 (hatched bar) ou 72 (dotted bar) hours after inoculum. A) number of infected cells in 100 counted cells; B) number of intracellular amastigotes in 100 counted cells; C) number of intracellular parasites per infected cell. *: Significant difference compared to the 24 hours of culture; #: Significant difference compared to the 48 hours of culture; a: significant difference compared to the strain Be-62; b: significant difference compared to the Be-78 parental strain. The data represent the mean of triplicates ± standard error.</p