<p>APPENDIX 2</p><p>Blood extraction procedure </p><p>Captured mammals were weighed under short-term isoflurane anaesthesia. Small mammal blood was withdrawn (0.2–1.0 ml) from a subset of the captured individuals by: (i) saphenous vein puncture for Octodon degus and Abrocoma bennetti with a 21G needle, (ii) maseterous vein puncture for P. darwini with a 21G needle, and (iii) jugular vein extraction for Abrothrix spp., Thylamys elegans and Oligoryzomys longicaudatus using a tuberculin syringe with a </p><p>25G needle and collected in vials (Johnson-Delaney 2006). The blood was immediately mixed with 6M Guanidine-HCl - 0.2M EDTA in a 1:1 proportion. Blood extraction procedure followed the international recommendations for mammals (Johnson-Delaney 2006), and was approved by the Ethical Committee of the Faculty of Science of the University of Chile, the </p><p>Chilean Agriculture and Livestock Bureau and the National Forest Corporation. All individuals were ear-tagged and released at the point of capture after recovery from anaesthesia.</p><p>Polymerase chain reaction (PCR) of faces and blood </p><p>The PCR method is highly specific (Junqueira et al. 2005), with an estimated sensitivity limit of 0.01 parasite equivalents/PCR assay (Arenas et al. 2012). The amplification reaction for faecal and blood samples was performed in triplicate with oligonucleotides 121 (5’-AAA TAA TGT ACG G (T/G) GAG ATG CAT GA-3’) and 122 (5'</p><p>GGG TTC GATTGG GGT TGG TGT-3), which anneal to the four conserved regions of the minicircles of T. cruzi, as described (Botto-Mahan et al. 2005b). A sample was considered positive when at least two of the three assays showed amplification. Samples with only one positive assay were considered doubtful and repeated three additional times. A sample was considered negative when none of the assays showed amplification. A sample of 5 μL of the elution of the extracted mammal blood or pre-boiled extract of triatomine faces was used as </p><p>DNA template in 50 μL of final volume. Each experiment included a blank that contained water instead of DNA and a positive control that contained purified kinetoplast DNA of T. cruzi. The minicircle hypervariable region PCR product of 330 bp was analysed by electrophoresis in a 2% agarose gel and visualized by ethidium bromide staining.</p><p>REFERENCES</p><p>Arenas M, Campos R, Coronado X, Ortiz S, Solari A (2012) Trypanosoma cruzi genotypes in</p><p> insect vectors and patients with Chagas of Chile studied by means of cytochrome b </p><p> gene sequencing, minicircle hybridization, and nuclear gene polymorphism. Vector-</p><p>Borne Zoonot 12:196-205.</p><p>Botto-Mahan C, Ortiz S, Rozas M, Cattan PE, Solari A (2005b) DNA evidence of </p><p>Trypanosoma cruzi in the Chilean wild vector Mepraia spinolai (Hemiptera : </p><p>Reduviidae). Mem I Oswaldo Cruz 100:237-239. doi:10.1590/S0074-</p><p>02762005000300003</p><p>Johnson-Delaney CA (2006) Common procedures in hedgehogs, prairie dogs, exotic rodents, </p><p> and companion marsupials. Vet Clin Exot Anim 9:415-435</p><p>Junqueira ACV, Degrave W, Brandao A (2005) Minicircle organization and diversity in </p><p>Trypanosoma cruzi populations. Trends Parasitol 21:270-272</p>