**2.2 Animals**

BALB/c, A and Swiss Webster female mice, 4–6 weeks old, were provided by Instituto de Ciência e Tecnologia em Biomodelos (ICTB - FIOCRUZ) and housed under pathogen-free conditions, controlled temperature, and food and water *ad libitum*.

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

### **2.3 Parasites**

*Trypanosoma cruzi* SC2005 (DTU Tc II), isolated from a case of oral acute Chagas' disease during an outbreak in Santa Catarina, Brazil [30, 31], was used in this study.

In experiments with outbred Swiss mice, epimastigote forms of *T. cruzi* SC2005 isolate were maintained in LIT medium for 30 days. Metacyclic trypomastigotes forms were quantified in a Neubauer chamber and used to infect VERO cells. The infected culture was maintained in RPMI medium, supplemented with 10% fetal bovine serum. Trypomastigotes derived from cell culture (TCC) were obtained 10 days after, by recovering parasites from the culture supernatant, and quantified in a Neubauer chamber prior to infecting mouse groups.

In experiments with inbred mice (A and BALB/c), epimastigote forms were maintained at 28°C for 21 days in LIT (Liver Infusion, Triptose) (AGM) culture medium to obtained metacyclic forms that were quantified in a Neubauer hemocytometer prior to infection.

### **2.4 Experimental design**

In order to investigate the influence of the route of infection on the course of *T. cruzi* SC2005 infection, Swiss mice were organized into three groups: group 1 (n = 30) mice intraperitoneally (IP) infected by 107 TCC forms of *T. cruzi* SC2005 strain/0.2 mL of RPMI medium; group 2 (n = 55) mice intragastrically infected by 107 TCC forms of *T. cruzi* SC2005 strain/0.1 mL of RPMI medium using a gavage needle (subjected to 4 h of fasting prior to infection), and group 3 (n = 15) normal uninfected animals (control group). Mice from each group (n = 3) were euthanized at 11 and 18 (IP-infected mice) and 26 and 33 (IG-infected mice) days after infection, and the esophagus, stomach, intestines, heart, thymus, liver, spleen, pancreas, kidney, adrenal gland, bladder, uterus, mesenteric lymph nodes, and brain were removed.

In experiments to investigate the host genetics influence on the course of *T. cruzi* SC2005 infection, A and BALB/c mice were intragastrically infected by 107 metacyclic trypomastigote forms of *T. cruzi* SC2005 strain/0.3 mL of LIT medium using a gavage needle. Prior to intragastric injection, the animals were submitted to 4 h of fasting. The animals were divided into four experimental groups: Group 1 (n = 70): A-infected mice; Group 2 (n = 70) BALB/c-infected, and Group 3 (n = 50) and Group 4 (n = 50) each one composed by uninfected A and BALB/c mice (control groups), respectively. Mice from each group (n = 6) were euthanized at 7-, 14-, 21-, and 40-day post-infection (dpi), and the blood, esophagus, stomach, gut, heart, and liver were removed.

#### **2.5 Parasitemia, mortality and leukometry**

Ten mice of each infected group were monitored daily from 5 to 50 dpi. Parasitaemia was determined as described by Pizzi and Prager [32]. Briefly, 5 μL of blood's tail vein was collected and placed under a cover slip (22 x 22 mm) and the number of parasites/mL of blood was estimated by counting 50 microscopic fields in a 400X magnification. Mice that did not develop parasitaemia were considered noninfected and excluded from the experiment.

Mortality rate was estimated to obtain the survivors percentage. Mean time of death was calculated following Liddell [33].

At the same time of parasitaemia evaluation, another 10 μL of blood's tail vein was collected and diluted in Turk's solution (1/20) [34] for white cells counting using a Neubauer chamber. Differential cell count was made in smears, after MayGrünwald– Giemsa staining, by counting 100 leukocytes/slide.
