PCR assay flowsheet¹ to identify <i>Trypanosoma cruzi</i> discrete typing units (DTUs).

<p>Directional arrows indicate assay order and stop signs denote when sufficient data was gathered to theoretically identify the DTU. The final assay (<i>GPI</i>) is included as a confirmatory step, but is not required for DTU identification. ¹Modified from Lewis et al. [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.ref037" target="_blank">37</a>] ²The large subunit rDNA assay is also referred to as the 24sα rRNA gene assay. ³An additional band of approximately 125bp may or may not be visible in combination with the 110bp band. ⁴Heat Shock Protein-60 (HSP60) results in an amplicon of 432-462bp, which upon RFLP with <i>Eco</i>V restriction enzyme yields the following patterns: 1 band (432–462), 2 bands (118–148 + 314), or 3 bands (118–148 + 314 + 432–462). ⁵This PCR used a pool of three primers to amplify a portion of the non-transcribed intergenic region of the tandemly repeated mini-exon gene. ⁶Glucose Phosphate Isomerase (GPI) results in an amplicon of approximately 1264bp, which upon RFLP with <i>Hha</i>I restriction enzyme yields the following patterns: 2 bands (447 + 817), 3 bands (253 + 447 + 490), or 4 bands (253 + 447 + 490 + 817). TcIV will display 2 or 3 bands for North American and South American strains, respectively.</p

Phylogeny for 26 <i>Trypanosoma cruzi</i> 350 bp RNA-Binding Protein-19 (<i>RB19</i>) sequences.

<p>Neighbor-Joining tree constructed in MEGA6 with evolutionary distances computed via Maximum Composite Likelihood. The numbers above the nodes represent bootstrap confidence levels for 2,000 replicates. Only values ≥ 50% are shown. The only TcI isolate not obtained in this study is represented by a square. Sequences obtained in this study are indicated by a triangle (Esc = Escondido, SoCal = Southern California, Vall = Vallecito). All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country origin indicated in parentheses. The scale bar indicates the number of nucleotide substitutions per site. Tree is outgroup rooted with <i>T</i>. <i>cruzi marinkellei</i> (TcMark CONTIG 1404).</p

Phylogeny for 35 <i>Trypanosoma cruzi</i> 1288 bp trypanothione reductase sequences.

<p>Depicted is the Neighbor-Joining (NJ) tree constructed in MEGA6 with evolutionary distances computed via the Maximum Composite Likelihood method and the scale bar indicating the number of nucleotide substitutions per site. The numbers above or below the nodes represent the bootstrap confidence levels for 2,000 NJ replicates (1^st value) and 500 Maximum Likelihood replicates (2^nd value presented at nodes with congruent topologies) run under the Kimura 2-parameter model for those values <b>≥</b> 50%. Sequences obtained in this study are indicated by the triangles (Esc = Escondido, SoCal = Southern California, Vall = Vallecito). All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country of origin indicated in parentheses. Tree is outgroup rooted with <i>T</i>. <i>cruzi marinkellei</i> (GenBank #AF359007).</p

Phylogeny for 62 <i>Trypanosoma cruzi</i> concatenated 786 bp cytochrome oxidase II-NADH 1 (<i>COII-ND1</i>) sequences.

<p>The TcI clade is condensed in this figure and contains the majority of the sequences obtained in this study (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.g005" target="_blank">Fig 5</a> for expanded version). Depicted is the Neighbor-Joining (NJ) tree constructed in MEGA6 with evolutionary distances computed via the Maximum Composite Likelihood method and the scale bar indicating the number of nucleotide substitutions per site. The numbers above or below the nodes represent the bootstrap confidence levels for 2,000 NJ replicates (1^st value) and 500 Maximum Likelihood replicates run under the Tamura 3-parameter model (due to slightly incongruent topology, ML bootstrap values are only shown at three nodes) for those values <b>≥</b> 50%. Only one sequence obtained in this study (Esc19 = Escondido 19) was grouped as TcIV based on mitochondrial gene sequences. All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country of origin indicated in parentheses. Tree is outgroup rooted with <i>T</i>. <i>cruzi marinkellei</i> (GenBank #AF359054).</p

TcI subtree represented as TcI on Fig 4 showing four distinct subclades.

<p>This subtree includes 46 <i>COII-ND1</i> concatenated sequences (786 bp). The scale bar indicates the number of nucleotide substitutions per site for the NJ tree. The numbers above or below the nodes represent the bootstrap confidence levels for 2,000 NJ replicates (1^st value) and 500 Maximum Likelihood replicates run under the Tamura 3-parameter model. Only bootstrap values <b>≥</b> 50% are shown. Sequences obtained in this study are indicated by the triangles (Esc = Escondido, SoCal = Southern California, Vall = Vallecito). Esc26 was grouped with TcI in this analysis but was grouped with TcIV in all other analyses. All other isolates represent published GenBank sequences as listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004291#pntd.0004291.s001" target="_blank">S1 Table</a> with their country of origin indicated in parentheses.</p

Performance characteristics of the local media surveillance (LMS) pilot by country.

<p>*Risk event: any report containing information on events, circumstances, or contexts that could increase potential transmission of a disease.</p><p>**Disease event: any report containing news of an actual disease (infectious or non-infectious).</p><p>Performance characteristics of the local media surveillance (LMS) pilot by country.</p

Health events identified through local media surveillance (LMS) and HealthMap’s digital disease surveillance over the 16-week evaluation period.

<p>Health events identified through local media surveillance (LMS) and HealthMap’s digital disease surveillance over the 16-week evaluation period.</p

Guidelines provided for the media source selection process.

<p>These general guidelines were provided at the onset of pilot implementation in each country to help the team members select the best weekly media sources for surveillance.</p

Changes in the serological status of wild dogs which were sampled twice, on dates 2.5–38 months apart.

<p>Animals which seroconverted were considered negative when first sampled, but positive subsequently, or <i>vice versa</i>. None of these animals was vaccinated against any pathogen.</p

Density of domestic dogs experienced by 57 African wild dogs, in 10 packs living mainly on either community lands or commercial ranches.

<p>Data show the mean (and SD) estimated density of domestic dogs at points where wild dogs were located by aerial radio-telemetry in the 12 months prior to sampling for pathogen exposure. Figures along the top of the graphs indicate the numbers of wild dogs sampled from each pack (over periods of 1–7 years).</p