APPENDIX 2

Blood extraction procedure

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 Small mammal blood was withdrawn (0.2–1.0 ml) from a subset of the captured individuals by: (i) saphenous vein puncture for Octodon degus and Abrocoma bennetti with a 21G needle, (ii)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

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

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.

Polymerase chain reaction (PCR) of faces and blood

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'

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

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.

REFERENCES

Arenas M, Campos R, Coronado X, Ortiz S, Solari A (2012) Trypanosoma cruzi genotypes in

insect vectors and patients with Chagas of Chile studied by means of cytochrome b

gene sequencing, minicircle hybridization, and nuclear gene polymorphism. Vector-

Borne Zoonot 12:196-205.

Botto-Mahan C, Ortiz S, Rozas M, Cattan PE, Solari A (2005b) DNA evidence of

Trypanosoma cruzi in the Chilean wild vector Mepraia spinolai (Hemiptera :

Reduviidae). Mem I Oswaldo Cruz 100:237-239. doi:10.1590/S0074-

02762005000300003

Johnson-Delaney CA (2006) Common procedures in hedgehogs, prairie dogs, exotic rodents,

and companion marsupials. Vet Clin Exot Anim 9:415-435

Junqueira ACV, Degrave W, Brandao A (2005) Minicircle organization and diversity in

Trypanosoma cruzi populations. Trends Parasitol 21:270-272