**2.6 Spleen index**

Spleen index was calculated to investigate reticuloendothelial stimulation. This index was calculated after evaluation of the relative spleen weight (spleen weight/mouse weight) [35].

## **2.7 Parasite load**

Fragments of heart recovered from infected mice were immediately frozen after euthanasia and stored at −70°C. Fragments were digested in 500 μL of lysis buffer (50 mM Tris, 10 mM NaCL, 5 mM EDTA, 0.5% SDS) containing proteinase K (20 mg/ml). DNA was extracted following a standard phenol/chloroform protocol [36]. DNA concentrations and purity were determined by reading A260 and A280 on a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

Parasite DNA was detected by SYBR Green qPCR assay using StepOnePlus Real-Time PCR System (Applied Biosystems). Primers targeting *T. cruzi* genomic DNA sequence (166 bp) Cruzi 1 (5'-ASTCGGCTGATCGTTTTCGA-3′) and Cruzi 2 (5'-AATTCCTCCAAGCAGCGGATA-3′) and Actb-actin, beta (b-actin) mouse gene (138 bp) (Forward 5'-AGAGGGAAATCGTGCGTGAC-3′; reverse 5'CAATAGTGATGACCTGGCCGT3') were used following previous reports [37, 38]. To monitor DNA integrity, variation in DNA yield, or the presence of potential inhibitors of PCR, Actb reference gene was used as a positive control. The reaction mixtures contained Power SYBR Green PCR Master Mix 2X (Applied Biosystems), 25 ng of DNA template, and 100 nM of b-actin or 300 nM of Cruzi1/ Cruzi2 primers in a final volume of 20 μL. PCR conditions were as follows: hold at 95°C for 10 min, 95°C for 15 s, and 58°C for 1 min (40x). Standard curves from axenic epimastigotes *T. cruzi* DNA (100 ng–1 pg) were generated. A melt curve analysis was performed on all reactions. The results were analyzed with the StepOne software v2.2.2 (Applied Biosystems).

## **2.8 Histopathology**

Following euthanasia, all removed organs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.45, at 4°C, for 72 h, cleaved and routinely processed paraffin embedded. Tissue sections (5 μm) were stained with Hematoxylin–Eosin (HE) (Sigma-Aldrich, Saint Louis, USA), Lennert's Giemsa, or Picro-Sirius red (Direct Red 80, Aldrich Milwaukee, WI 53233, USA) techniques. The presence of inflammatory infiltrates was classified as (−) without infiltrates, (+) very mild lesion areas, (++) mild lesion areas, (+++) moderate areas of infiltrates, (+++) severe areas of infiltrates, (++++) very severe areas of infiltrates, following described by Barreto-de-Albuquerque et al. [39], and arbitrary values from 0 to 5 were attributed to it. Tissues were analyzed and photographed under light microscopy (Zeiss, Axioplan 2, with Axiovision LE64 photomicrograph equipment).

*How Do Mouse Strains and Inoculation Routes Influence the Course of Experimental… DOI: http://dx.doi.org/10.5772/intechopen.104461*
