**3.1 Route of infection and host genetic background influence on the parasitaemia and mortality after** *T. cruzi* **SC2005 infection**

Several studies have been showing that the course and severity of Chagas' disease depends on varied factors, among them are the route of infection and host genetic background [25].

The most frequent route of Chagas' transmission is currently by ingestion of contaminated food and beverages, especially in Brazil and some other endemic countries [19, 40–42]. Infection by oral route usually leads to a more severe acute disease than vector-borne infection. The amount of metacyclic trypomastigotes contained in a triatomine crushed in food or beverage may be more than 100 times higher than what is typically found on its feces [43]. Furthermore, the digestive mucosa represents an extensive gateway, containing different molecules that serve as attachment points to the parasite [44]. Therefore, orally infected patients tend to experience a more severe acute phase, with more rapid progression to long-term cardiac or gastrointestinal dysfunction and higher mortality [43]. In the experimental model, intraperitoneal inoculation is the most common route of infection, but it does not mimic any natural infection. It delivers elevated loads of trypomastigotes directly in the peritoneum, bypassing all natural barriers in the skin and mucosa. When mucosa and systemic *T. cruzi* infection were compared, distinct disease patterns can be observed. Several studies in mice showed that intraperitoneal infection induces higher parasitemia and mortality than intragastric (IG) or oral infection with the same inoculum [39, 45, 46]. Marsden [47] showed that systemic infections (intraperitoneal or intravenous) promote higher

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

infection rates (67–100%) and mortality than mucosal (oral, intragastric, intrarectal, genitalia, or conjunctival infection) (17–67%) in mice infected by the Peruvian strain. In the present study, similar results were obtained when outbred Swiss mice were infected IG or IP with TCC forms of SC2005 *T. cruzi* strain. IP infection was able to infect 100% of animals, while just 36% of IG-infected mice developed parasitaemia. Moreover, IP-infected mice showed earlier (10th and 13th days) and higher parasitaemia peaks (2.9 and 4.3 X 106 parasites/mL, respectively) than those observed in the IG-infected animals, which presented peaks on the 13th and 18th dpi with 0.9 and 1.7 X 106 parasites/mL, respectively (**Figure 1A**). Furthermore, IP-infected animals died earlier (mean time of death of 16.13 ± 0.8 days), and its mortality rate was 80%. On the other hand, IG-infected mice died later (22.67 ± 2.0), and the mortality rate was around 30% (**Figure 1B**). Different infection routes submit parasites to different barriers in order to infect the host. Crossing of these barriers may explain the differences observed in course and intensity of the parasitaemia. Nevertheless, independent of the route of infection, parasites were able to make their way to the heart, which showed amastigote nests after infection by both routes (**Figure 1C** and **D**).

The success of *T. cruzi* infection and the level of parasitaemia after oral contamination are mainly determined by the magnitude of the mucosal immune response developed [48], which depends on the interaction between both parasite and host genetics [49, 50]. Studies of genetic susceptibility to Chagas' disease are scarce and its contribution to disease pathology is still unsolved. Inbred mice from different genetic backgrounds have been used to assess the influence of host genetics to several pathogens. Studies using inbred mouse strains infected by *T. cruzi* showed different profiles of response to infection and degrees of susceptibility in the hosts, with differences in mortality rate, cytokine production, inflammatory infiltrate, and parasite load [51–53]. C57BL/10 mice infected with *T. cruzi* SC2005 strain showed lower parasitaemia and mortality rate, while CBA-infected mice showed high parasitaemia and mortality rate [52]. Other studies using different inbred mouse strains (A/J, BALB/c, C3H/HePas, C57BL/6, and DBA mice) infected with *T. cruzi* Y also showed differences in mortality rate and parasitaemia. C57BL/6 mice were less susceptible to infection, while A/J was the most susceptible strain, showing the highest parasitemia and mortality rate [51]. In this work, we also observed significant differences in both parasite load and mortality rate between two mouse strains intragastrically infected with *T. cruzi* SC2005. The A mice were less susceptible to infection, showing lower parasitaemia (**Figure 2A**), parasite load in the heart (**Figure 2B**), and mortality rates (**Figure 2C**). Although these animals presented an earlier mortality (23 dpi), only 10% of the infected mice died. On the other hand, BALB/c mice presented a higher mortality rate (25%), but a later mean time of death (28,4 dpi) (**Figure 2C**). Since the same protocol and *T. cruzi* strain were used to infect both mouse lineages, we can suggest that the differences in the parasite load and mortality are influenced by immunological response developed by each mouse strain, which is determined by their genetic backgrounds. Nonetheless, the actual basis for that difference is unknown. BALB/c mice have been described to carry the susceptible genotype for the *Scl11c1* gene, a divalent ion transporter present on monocytes, macrophages, NK cells, and ɣδ T cells, which have roles in phagosome maturation, cell activation, and IFN production, rendering hosts susceptible to several intracellular pathogens, such as *Leishmania donovani*, *Salmonella typhimurium*, and *Mycobacterium sp* [54–56]. The A/J strain, on the other side, carries the resistant genotype [57, 58], being less susceptible to those pathogens. Nevertheless, this locus alone cannot explain host resistance to all pathogens, since BALB/c and A/J mice are both highly susceptible


#### **Figure 1.**

*Influence of the route of infection on parasitaemia, mortality, and heart histopathology. Swiss Webster outbred mice were infected either intraperitoneally (IP) or intragastrically (IG) with 107 TCC of* Trypanosoma cruzi *SC2005 strain. Parasitaemia (A) shows an early and strong increase of parasites in the blood in IP-infected mice, which leads to an earlier time to death (B). IG-infected mice present a weak rate of infection (B) and a later and lighter parasitaemia (A). Nevertheless, both routes led to a colonization of the heart myocardium, which can be seen after 18 days on IP-infected mice (C) and 26 days on IG-infected animals (D). Hematoxylin and eosin. Arrows show parasite nests. Two-way ANOVA followed by Sidak's multiple comparison test. \*\*\* = p < 0.001.*

to *Staphylococcus aureus* infection [56] and BALB/c is resistant to hepatitis caused by Rift Valley Virus [57]. Susceptibility to infection is usually complex and dependent on multiple loci, which cannot be easily identified, but mouse genetics is a powerful tool to study host genetics.

In our study, BALB/c and A mice presented parasites nest on the heart (**Figure 2D** and **E**). Cardiac damage in acute Chagas disease is closely related to cases of death [5, 59]. The highest number of deaths observed in BALB/c *T. cruzi* SC2005 infected mice may be related to the extensive damage of the heart, caused not only by the parasite itself, but also to the inflammatory response against the parasite in this

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

#### **Figure 2.**

*Influence of the mouse strain on the parasite load. Inbred mice from A and BALB/c genetic backgrounds were infected intragastrically (IG) with 107 metacyclic forms of* Trypanosoma cruzi *SC2005 strain. BALB/c presented higher parasitaemia (A), higher parasite load on the heart, measured by qPCR (B) and higher mortality rate (C) than A mice. Histopathology from heart of BALB/c animals shows inflammatory infiltration (D) and parasite nests (E; arrows). Hematoxylin and eosin. Two-way ANOVA followed by Sidak's multiple comparison test. \* = p < 0.05; \*\*\*\* = p < 0.0001.*

organ, once this mouse strain showed an inflammation on the heart more extensive and intense than A mice. A wide genomic association study carried out with patients from Colombia, Bolivia, and Argentina has identified a QTL in chromosome 11 which is associated with the development of Chagasic cardiomyopathy [28]. The QTL seems to correspond to a methylation site at the CCDC88B gene, which is involved in the inflammatory response [60].

### **3.2** *T. cruzi* **SC2005 infection induces hematological alterations**

In this work, hypochromic anemia was the main hematological alteration found after *T. cruzi* SC2005 intragastric infection. BALB/c mice showed hypochromic anemia earlier (14 dpi) than A-infected mice (21 dpi) (**Figure 3**). Anemia is a common

hematological alteration observed in acute Chagas' disease, as described by Chagas in patients infected by *T. cruzi* [8]. In experimental infections, this hematological alteration also has been shown in different mouse strains [61]. However, the mechanisms responsible for this alteration are still unsolved. In acute *T. cruzi* infection, the lethality is associated with the reduced number of blood cells and the impaired bone marrow function [62]. All these alterations may be influenced by cytokine secretion and parasite or cell-dependent cytotoxicity in the blood and bone marrow [63].

#### **Figure 3.**

*Hematological analysis. A-E inbred mice from A and BALB/c genetic backgrounds were infected intragastrically (lg) with 107 metacyclic forms of* Trypanosoma cruzi *SC2005 strain. BALB/c mice shows a reduction in red blood cells count (RBC; A), hematocrit (Hct; B), and hemoglubin (Hgb; C). The mean corpuscular volume (MCV) of BALB/c red blood cells also presented a slight reduction (D). Extracellular hematopoiesis could be observed in the liver of BALB/c mice (E) but also in outbred Swiss mice intraperitoneally infected with 107 TCC of*  T. cruzi *SC2005 strain (F), where immature cells are also observed (G). E: Giemsa; F and G: Hematoxylin and eosin. Two-way ANOVA followed by Sidak's multiple comparison test. \* = p < 0.05; \*\*p = <0.01; \*\*\* = p < 0.001; \*\*\*\* = p < 0.0001 in comparison with normal mice from the same background. # = p < 0.05; ## = p < 0.01; ### = p < 0.001; #### = p < 0.0001, comparing infected mice from different genetic backgrounds.*

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

A reduced life span or sequestration of RBC by autoantibodies or other mechanisms have been described as a factor that can contribute to anemia in protozoan and viral infections [64–69]. The average life span of a red blood cell is 40 days in a normal mouse [70]. In this study, *T. cruzi* infection reduced the life span of RBC contributing to anemia, once both mouse-infected strains showed an anemia earlier to this period, between 14 and 21 dpi (**Figure 3**).

Bone marrow suppression is related with the activity of several cytokines, and among them are TNF-α and IFN-γ [71–74]. It was shown that an excessive production of TNF-α and IFN-γ promotes damage in hematopoiesis in mice infected with lymphocytic choriomeningitis virus (LCMV) [71]. In studies with malaria, TNF-α has been described as an important anemia mediator [64]. In previous studies, inhibitory effects of TNF-α on erythropoiesis have been demonstrated [74, 75]. This inhibitory effect was also observed during acute infection by *T. cruzi*, in which the production of TNF-α by activated macrophages was correlated with a decrease in erythropoiesis [63]. In our study, the results indicate that the anemia observed after *T. cruzi* infection can be associated with a depressed bone marrow function induced by TNF-α and IFN-γ, once both infected mouse strains produced high levels of TNF-α and IFN-γ, 14 days after infection (see below), correlating with the decrease of RBC and Hgb (**Figure 3A** and **C**).

One consequence of the depressed bone marrow function, characterized by the poor quality or insufficiency of the blood elements production, is extramedullary hematopoiesis (EMH). EMH can occur in adult mouse livers [65, 66] under myelosuppression by various pathological lesions, including hemoglobinopathies, most commonly sickle cell anemia and thalassemia [67, 68]. In our study, we observed the occurrence of extramedullary hematopoiesis in the livers of *T. cruzi*-infected animals at 14 and 21 dpi, characterized by the presence of megakaryocytes, immature hematopoietic cells, and mitotic cells (**Figure 3E, F** and **G**). Altogether, these findings corroborate the hypothesis that *T. cruzi* SC2005 infection causing an impairment in bone marrow function, induced by TNF-α and IFN-γ, which lead to the occurrence of anemia and extramedullary hematopoiesis in mice livers as a compensatory mechanism.

Leukocytosis is another hematological alteration described after *T. cruzi* infection. A systematic review analyzing 31 articles up to 2016 showed that half of them described anemia in infected mammals and 68.2% described leukocytosis [76]. According to Tribullatti et al. [77], molecules such as chemokines, cytokines, antibodies, and nitric oxide produced during *T. cruzi* infection, together with molecules produced by the parasite itself, lead to hematological alterations in infected animals. In this study, a significant leukocytosis was observed in A and BALB/c mouse strains 21 and 40 days after *T. cruzi* SC2005 intragastric infection (**Figure 4A** and **B**). This leukocytosis was associated with the parasitemia levels and characterized by monocytosis and lymphocytosis, as well as by the presence of a lymphocytic atypia. When comparing infection routes, both IP- and IG-infected animals presented an increased number of leukocytes at the same time when parasitaemia increased. Nevertheless, leukocytosis is much higher in IG-infected mice (p < 0.0001, comparing leukocytosis peak, day 12 for IP- and 18 for IG-infected mice), even with a lower parasitaemia. In both cases, leukocytosis is caused mainly by an increase of lymphocytes, although neutrophils also follow the increase (**Figure 4C**-**F**). Several works previously reported alterations in leukocyte counts associated with parasitemia levels in different experimental models. Cynomolgus macaque (*Macaca fascicularis*) naturally infected by *T. cruzi* [78] and Rhesus monkeys experimentally infected with *T. cruzi* Colombian

#### **Figure 4.**

*White blood cell count analysis. Inbred mice from A (A) and BALB/c (B) genetic backgrounds intragastrically infected with 107 metacyclic forms of* Trypanosoma cruzi *SC2005 strain presented leukocytosis associated with parasitaemia. C-F. Swiss Webster outbred mice were infected either intraperitoneally or intragastrically with 107 TCC of* T. cruzi *SC2005 strain. Mice infected intragastrically show an increase in the total leukocytes, lymphocytes (D), and neutrophils (F) at the same time of the parasitemic peak.*

strain [79] showed a positive correlation between leukocytosis, lymphocytosis, and parasitemia peaks. The same correlation was found in Beagle dogs infected by different *T. cruzi* strains [80, 81]. On the other hand, other authors obtained the contradictory results during experimental murine infection with *T. cruzi* CL strain in C3H mice, showing an exponential growth of parasites accompanied by leukopenia in these animals [82]. Such dissimilarity in experimental data suggests that both the host genetic background and the *T. cruzi* strain may influence the hematological alterations occurring after infection.

### **3.3 Immune response in** *T. cruzi* **SC2005-infected mice**

Recognition of *T. cruzi* by macrophages and dendritic cells leads to phagocytosis and elicits a predominately T helper type 1 (Th1) response with the production of pro-inflammatory cytokines (i.e., IFN-γ, IL-2, IL-6, IL-12, and TNF-α) [83–85]. This promotes an antiparasitic response, with differentiation and proliferation of Th1 CD4+ cells, activation of CD8+ T cells, and macrophages [86]. The recognition of *T. cruzi*-infected cells and effective control of infection during the acute and chronic phases are mainly related to the action of effector CD8+ T cells [48, 79, 87]. More severe clinical disease is associated with reduced CD8+ T cell responses and the boost of CD8+ T cell response after treatment improves the clinical outcome [88, 89]. However, studies indicate that, in the absence of CD4+ T cells, CD8+ T lymphocytes fail to restrain the parasite growth [90], because CD4+ T lymphocytes are responsible for promoting the macrophages activation and CD8+ T and B cells proliferation. Therefore, its deficiency leads to an overall reduction of host immune response and a consequent increase in tissue parasitism [91].

In our study, the frequencies of CD4<sup>+</sup> , CD8<sup>+</sup> , CD4<sup>+</sup> /CD8<sup>+</sup> T cells and B cells in different organs varied according to the route of infection, and mouse strain. After intraperitoneal infection by *T. cruzi* SC2005 strain, Swiss mice presented an increase in CD4<sup>+</sup> T cells frequency in the spleen at 18 dpi. On the other hand, IG-infected mice showed an increase in the frequencies of CD8<sup>+</sup> T cells 26 and 33 dpi (**Figure 5A**). The increase of these cells corroborates with the increase in spleen weight (**Figure 6A**), caused by the intense production of cells, as shown by the hyperplasia of the germinal center induced by both infection routes in this organ (**Figure 6B** and **C**). An increase in the cellularity and size of spleen, caused by the amplification of T and B lymphocytes polyclonal activation, has been previously described during *T. cruzi* infection [92].

In the blood, IG-infected mice showed a reduction of CD4<sup>+</sup> T cells frequency (26 dpi) and an increase of CD8+ T cells 26 and 33 days after infection (**Figure 5B**). Meanwhile, the analysis of lymph nodes showed a decrease of CD4+ T cell frequency on both infected groups: IP-infected mice at 11 dpi and IG-infected mice 26 and 33 dpi (**Figure 5C**). The variation in the T cells frequency in the mesenteric lymph nodes is caused by depletion of lymphocytes and increased apoptosis rates, which may be related to the production of different cytokines [92].

These results show a fluctuation in the percentage of lymphocytes in the analyzed tissues. This variation demonstrates the differentiated role of each compartment and a different response profile with a specific and coordinated response of these sites against the parasite [93]. Besides, we demonstrated the influence of infection route on the lymphocyte's frequency and magnitude, once *T. cruzi* IG-infected mice showed a higher expansion of CD8<sup>+</sup> T cells in the spleen and blood.

The differences in expansion and distribution of T and B cell frequencies in the blood, liver, and heart of A and BALB/c mice IG infected by *T. cruzi* SC2005 were also demonstrated in this work. SC2005 *T. cruzi* IG infection induced an increase of CD8+ T and CD4+ /CD8+ T lymphocytes in all analyzed organs on both mouse strains, although in A mice the increase occurred earlier than in BALB/c mice. The infection also induced a decrease in CD4<sup>+</sup> T cell and B lymphocyte frequencies in the heart and blood of infected animals (**Figure 7**). It is known that in both acute and chronic

#### **Figure 5.**

*T cells present in the inflammatory infiltrate. Swiss Webster outbred mice were infected either intraperitoneally (IP) or intragastrically (IG) with 107 TCC of* Trypanosoma cruzi *SC2005 strain. Intragastrically-infected mice show an increase of T CD8+ cells in the spleen (A) and blood (B), but not in the draining lymph node (C). IP = intraperitoneally infected; IG = intragastrically infected. Two-way ANOVA followed by Sidak's multiple comparison test, in comparison to non-infected normal mice. \* = p < 0.05; \*\*\* = p < 0.001; \*\*\*\* = p < 0.0001.*

*T. cruzi* infections, the increase of CD8+ T lymphocytes is common [69]. Increased levels of these cells have been described as Chagas disease biomarkers in humans and monkeys naturally infected by *T. cruzi* [70]. CD8<sup>+</sup> T cell type-1 (Tc1) subsets are the main cause of *T. cruzi* death through the production of both IFN-γ and TNF-α [94, 95], contributing to the control of parasitaemia levels [91, 96, 97]. In this study, BALB/c and A mice infected by *T. cruzi* SC2005 presented lymphocytosis (**Figure 4A** and **B**) and an increase of CD8<sup>+</sup> T cells in the blood at the same time of the parasitaemia peak (21 dpi), remaining until the end of the experiment (40 dpi), when the parasite load was very low (**Figure 2A**). These results reinforce the previous *How Do Mouse Strains and Inoculation Routes Influence the Course of Experimental… DOI: http://dx.doi.org/10.5772/intechopen.104461*

#### **Figure 6.**

*Cell proliferation in the spleen. Swiss Webster outbred mice were infected either intraperitoneally or intragastrically with 107 TCC of* Trypanosoma cruzi *SC2005 strain. All mice present a higher spleen index (A) than normal mice, showing cell proliferation is occurring on the organ, which is corroborated with histopathology image from intraperitoneally (B) and intragastrically (C) infected mice. Hematoxylin and eosin. One-way ANOVA followed by Sidak's multiple comparison test. \* = p < 0.05; \*\*\*\* = p < 0.0001.*

observations about the role of CD8+ T cells in the control of parasitaemia levels and its probable cytotoxic activity against the parasite, as reported previously [78].

CD4<sup>+</sup> /CD8+ double-positive T cells, under normal conditions, are found in the thymus, where they undergo differentiation into mature CD4+ and CD8+ T cells. During *T. cruzi* infection, there is a significant impairment of this organ, due to a deregulated cascade of proinflammatory cytokines. This impairment leads to cell maturation in extrathymic organs such as the bone marrow and liver [98, 99]. In this study, the increase of the CD4+ /CD8+ T double-positive cells frequency in the heart, liver, and blood indicates an impairment of the thymus and consequent liberation of immature cells in the circulation or the occurrence of extracellular hematopoiesis in the liver (**Figure 3E**).

In *T. cruzi* infection, the clearance of blood trypomastigotes occurs in the liver [100], regardless of the gateway. The liver is the main organ involved in the defense against disseminating blood pathogens, being critical to host immunity and survival [101, 102]. In this work, an increase of both CD8+ T cells and CD19<sup>+</sup> B cells was observed in the hepatic parenchyma (**Figure 7C**). B cells act as antigen-presenting

#### **Figure 7.**

*Influence of the mouse strain on cell recruitment. Inbred mice from A and BALB/c genetic backgrounds were infected intragastrically (IG) with 107 metacyclic forms of* Trypanosoma cruzi *SC2005 strain.* T. cruzi *Infection caused an increase of CD8+ cells on the heart (A), blood (B) and liver (C) of infected mice. However, BALB/c CD8+ cells increased from 21 days after infection, whereas at 14 days after infection A mice already presented higher frequencies of CD8+ cells. The same happened with CD4+/CD8+ in mice blood. (B) CD19+ cell frequency was reduced in the blood from both mouse strains. Lines show normal mice mean frequencies. Two-way ANOVA followed by Sidak's multiple comparison test in comparison with normal mice from the same background. \* = p < 0.05; \*\* = p < 0.01; \*\*\* = p < 0.001; \*\*\*\* = p < 0.0001.*

cells (APC), in the secretion of antibodies, and in the activation of CD8<sup>+</sup> T cells [103]. Besides, they have been implicated in the mobilization of inflammatory cells to the tissues and are fundamental to the control of parasite growth, by triggering a Th1 response [90, 104, 105]. The observation of these cells in the liver suggests the role of CD19<sup>+</sup> cells as antigen-present cells (APC). A mice showed an earlier increase of these *How Do Mouse Strains and Inoculation Routes Influence the Course of Experimental… DOI: http://dx.doi.org/10.5772/intechopen.104461*

cell frequencies (14 dpi) than BALB/c-infected mice (21 dpi) (**Figure 7C**). The early presence of B and CD8<sup>+</sup> T cells in the liver of A mice indicates the importance of this organ in parasite clearance and may explain why this mouse strain has lower parasite load and mortality.

Redistribution and circulation of lymphocyte subtypes to other sites are modulated by the local and systemic immune system [106]. This regulation is affected by the infection and often related to the severity of the clinical manifestations.

Several murine studies have described the importance of proinflammatory cytokines during *T. cruzi* infection. C57BL/6 IL-17A knockout (IL-17A −/−) mice infected by *T. cruzi* Tulahuén strain showed a reduced production of IFN-γ, IL-6, and TNF-α cytokines, which was related to a more severe parasitemia and mortality, than observed in wild-type mice [107]. On the other hand, an improvement of the mice resistance to parasite growth was observed when IL-18 or 5-lipoxygenase was inhibited, generating an increase in the IL-12, IFN-γ, IL-1β, and IL-6 levels during the acute phase of disease [108, 109]. Studies *in vitro*, using PBMC infected by *T. cruzi* Tulahuén strain, indicate that IL-6 improves the survival and effector functions of cytotoxic cells [110]. These data reveal the important role of these cytokines in the control of *T. cruzi* infection and host mortality. In the present work, both infected mouse strains produced a similar pattern of TNF-α, IFN-γ, and IL-6 production, but A-infected mice showed an earlier increase in the production of these cytokines, when compared to BALB/c mice (**Figure 8**).

### **3.4 Histopathological alterations caused by SC2005** *T. cruzi* **infection**

After infection, *T. cruzi* can be detected in several organs/tissues and induce an inflammatory response, which can be found even where the parasite is not detected [111, 112]. The preferential tropism, as well as distinct local and systemic immune responses can be influenced by different transmission routes of *T. cruzi* [39, 47, 112, 113] as well as by the host genetic background [114].

In this study, we observed differences in the parasite load and inflammatory infiltrate intensity depending on the inoculation route used. IP infection showed an higher tissue colonization by SC2005 *T. cruzi* and a more intense inflammatory infiltrate than in IG infection. IP-infected mice showed a moderate-to-intense diffuse mononuclear inflammatory infiltrate (composed mainly by monocytes and lymphocytes) in the esophagus, stomach, intestine, heart, liver, pancreas, adrenal gland, bladder, uterus, trachea, and adipose tissue (**Figure 9**). Parasite nests were observed in the stomach (**Figure 9A**), esophagus (**Figure 9B**), intestine, heart, pancreas, bladder, and adipose tissue. Mast cells were observed only at 18 dpi in inflammatory infiltrates of the heart, stomach, and adipose tissue (**Figure 9D**). Severe pancreatitis with focal necrosis were also observed (**Figure 9E** and **F**) and the liver of the infected mice showed immature cells, megakaryocytes, and dividing cells (**Figure 3F** and **G**). In addition, a redistribution and increase of collagen fibers, associated with inflammatory infiltrates, was observed in the esophagus, heart (**Figure 9G**), stomach (**Figure 9H**), bladder, and uterus. Thymus and brain showed no changes in these animals.

On the other hand, IG infection induced a moderate-to-intense diffuse inflammatory infiltrate essentially lymphomonocytic in the esophagus, stomach, liver, kidney, bladder, uterus, brain, intestine, heart, pancreas, and adipose tissue (**Figure 10**). Mast cells were observed in the inflammatory infiltrates of the adipose tissue, bladder, and stomach (**Figure 10A**). Parasite nests were observed in lower numbers than IP-infected mice only in stomach, heart, bladder (**Figure 10D**), and adipose tissue. Thymus

#### **Figure 8.**

*Cytokine production by mice from different strains. Inbred mice from BALB/c (A) and A (B) genetic backgrounds were infected intragastrically (IG) with 107 metacyclic forms of* Trypanosoma cruzi *SC2005 strain. Both strain presented similar cytokine production, although it starts earlier in A mice. Two-way ANOVA followed by Sidak's multiple comparison test. ## = p < 0.01, in comparison with the other mouse strain.*

showed no changes in these animals. A common alteration observed in both IP- and IG-infected animals was the germinal centers hyperplasia in spleen and lymph nodes (**Figure 6C**), as well as omental and mesenteric milky spots were activated (**Figure 10C**), and myeloid cells are present. In addition, the liver of the infected mice showed immature cells, megakaryocytes, and dividing cells.

Both infected mice showed a redistribution and an increase of collagen fibers, associated with inflammatory infiltrates and a decrease of these infiltrates with the course of infection, with exception of the heart, where there is an increase of these infiltrates in the later times (IP-18 dpi and IG-33 dpi). These inflammatory infiltrates were preferentially located in the muscle layer of the organs. In the heart, they were preferentially located in the atria, while parasites were found in the heart ventricles. IP-infected mice showed an increase in heart parasitism at 18 dpi. On the contrary, IG-infected mice showed scarce parasite nests in this organ at 33 dpi and a much larger inflammatory infiltrate (essentially lymphoid) than observed in IP-infected mice (**Figure 1C** and **D**).

Similar to the observed Swiss mice infected by IG or IP routes, A and BALB/c mice IG infected by SC2005 *T. cruzi* strain showed immature cells and megakaryocytes in the liver (**Figure 3E**); and mononuclear inflammatory infiltrates associated with the increase of collagen fibers in several tissues (**Figure 11**). Both mouse strains presented inflammatory infiltrates in several organs, but in BALB/c the stomach (21 and 40 dpi), heart (14 and 21 dpi) (**Figure 2D**), and liver (7 dpi) were more inflamed. On the other hand, the esophagus of A mice presented an intense inflammatory infiltrate, whereas BALB/c's did not show any alterations. Neither mouse strains present histopathological changes in the gut (**Figure 11A**).

As it can be observed, the SC2005 *T. cruzi* infection induced similar alterations independent of the route of infection (IP or IG) and the mouse strain (BALB/c or A). The histopathological analysis showed a mononuclear infiltrate mainly located in the muscular layers, which was associated with neoformation and remodeling of collagen fibers in different organs (**Figure 11D** and **E**). Studies with *T. cruzi* strains

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

#### **Figure 9.**

*Histopathological alterations in intragastrically-infected mice. Swiss Webster outbred mice were intraperitoneally (IP) infected with 107 TCC of* Trypanosoma cruzi *SC2005 strain. Inflammatory infiltration and parasite nests (arrows) can be observed in mice stomach (A) and esophagus (B). Different other tissues also present intense inflammation, including supra renal (C) and adipocytes, in which we can observe mast cells (arrow head) (D). IP infected mice presented severe pancreatite (E and F). Collagen fibers can be observed in heart (G) and stomach (H) of mice. Hematoxylin and eosin (A-F) and Picrus Sirius red in polarized light (G-H).*

belonging to the biodemes type II and III showed the same histopathological patterns of localization of inflammatory infiltration [5]. The deposition of collagen and the tissue remodeling during *T. cruzi* infection have already been described in several studies [115–117]. In the heart, amastigote nests were present more frequently in the ventricles than in the atria. However, the atria presented a more intense inflammatory infiltration than ventricles. These findings suggest that the inflammatory

#### **Figure 10.**

*Histopathological alterations in intraperitoneal infected mice. Swiss Webster outbred mice were intragastrically (IG) infected with 107 TCC of* Trypanosoma cruzi *SC2005 strain. Moderate inflammatory infiltrate are present in different tissues such as stomach (A), esophagus (B), intestine (C), bladder (D), uterus (E), and meninges (F). Several mast cells can be observed in mice stomach (A). Activated milk spots are present in the intestine (C) and small parasite nests were observed in the bladder (arrow) (D). Hematoxylin and eosin.*

response against the parasite first occurs in the auricular tissue and only later reaches the ventricular tissue. It is interesting to note that Quijano-Hernández et al. in 2012 [118] observed the same histopathological patterns in infected dogs. Cardiac damages are closely related to cases of death in acute Chagas disease [5, 59]. In this study, a larger cardiac involvement and a higher parasite load were observed in the hearts of BALB/c-infected mice. The extensive damage of the heart plus the large number of parasite DNA and a late CD8<sup>+</sup> T cell response, corroborate to the highest number of deaths in this group.

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

#### **Figure 11.**

*Histopathological alterations in intragastrically-infected BALB/c mice. Inbred BALB/c mice were infected intragastrically (IG) with 107 metacyclic forms of* Trypanosoma cruzi *SC2005 strain. Inflammatory infiltration was quantified in various organs (A) inflammation can be observed in mice stomach (B) and liver (C). Alteration of collagen fibers are also observed in heart (D), and stomach (E). Hematoxylin and eosin (B-C) and Picrus Sirius red (D-E).*

The presence of immature cells and megakaryocytes in the liver was also a common finding in all infected mice. This finding suggests that extramedullary hematopoiesis was occurring in this organ. During the acute phase of *T. cruzi* infection, Marcondes et al. in 2000 [82] demonstrated alterations in blood cell counts associated with bone marrow suppression and anemia, which explain the occurrence of extramedullary hematopoiesis.
