Oral Versus Intragastric Inoculation: Similar Pathways of Trypanosoma cruzi Experimental Infection? From Target Tissues, Parasite Evasion, and Immune Response

Abstract

Currently, oral infection is the most frequent transmission mechanism of Chagas disease in Brazil and others Latin American countries. This transmission pathway presents increased mortality rate in the first 2 weeks, which is higher than the calculated mortality after the biting of infected insect vectors. Thus, the oral route of Trypanosoma cruzi infection, and the consequences in the host must be taken into account when thinking on the mechanisms underlying the natural history of the disease. Distinct routes of parasite entry may differentially affect immune circuits, stimulating regional immune responses that impact on the overall profile of the host protective immunity. Experimental studies related to oral infection usually comprise inoculation in the mouth (oral infection, OI) or gavage (gastrointestinal infection, GI), being often considered as similar routes of infection. Hence, establishing a relationship between the inoculation site (OI or GI) with disease progression and the mounting of T. cruzi-specific regional immune responses is an important issue to be considered. Here, we provide a discussion on studies performed in OI and GI in experimental models of acute infections, including T. cruzi infection.

Keywords: T cell activation; Trypanosoma cruzi; immune response; intragastric infection; oral cavity.

Figure 1

Severity and target tissues during acute phase of Trypanosoma cruzi orally infected mice. (A) Male BALB/c mice were infected with 5 × 10⁴ tissue culture-derived trypomastigotes forms of T. cruzi (Tulahuén strain) through gavage [gastrointestinal infection (GI)] or oral cavity (OI). Parasitemia (mean and SEM) was assessed during the acute phase and expressed as ln parasites per milliliter for statistical analysis. Parasites were counted by light microscopy, and parasitemia calculated by the Brenner method. Parasitemia comparisons were performed at different days post-infection (dpi), Kruskal–Wallis, Dunn’s post-test (until 15 dpi), and one-tailed Mann–Whitney (after 15 dpi) tests were used. (A) n: GI, 3 dpi = 7; 7 dpi = 22; 9 dpi = 29; 12 dpi = 17; 15 dpi = 45; 17 dpi = 10; 21 dpi = 24; 25 dpi = 16; 29 dpi = 11 and OI, 3 dpi = 4; 7 dpi = 9; 9 dpi = 14; 12 dpi = 22; 15 dpi = 40; 17 dpi = 12; 21 dpi = 14; 25 dpi = 8; 29 dpi = 6. Lower numbers represent early stages, when parasitemia was still undetectable and final stages, when mortality rates were too high. (B) Cytokine analysis in GI and OI mice. Male BALB/c mice were infected with 5 × 10⁴ tissue culture-derived trypomastigotes forms of T. cruzi (Tulahuén strain) through gavage (GI) or within oral cavity (OI). In the course of acute infection, serum was isolated and levels of cytokines (IFN-γ, TNF, IL-17, IL-10, and TGF-β) were quantified in uninfected control and infected mice by a multiplex analysis. The results are expressed as the mean values (±SEM) for each group/day post-infection. n: IFN-γ, uninfected (0) = 12; 3 dpi GI = 11, OI = 5; 9 dpi GI = 8, OI = 5; 12 dpi GI = 9, OI = 4; 17 dpi GI = 4, OI = 6. TNF, uninfected (0) = 11; 3 dpi GI = 10, OI = 10; 9, 12 dpi, GI = 3, OI = 3; 17 dpi, GI = 6, OI = 11. IL-17, uninfected (0) = 12; 3 dpi, GI = 10, OI = 10; 9 dpi, GI = 3, OI = 3; 12 dpi, GI = 5, OI = 5; 17 dpi, GI = 6, OI = 14. TGF-β, uninfected (0) = 6; 3 dpi, GI = 4, OI = 4; 9 dpi, GI = 5, OI = 5; 12 dpi, GI = 5, OI = 4; 17 dpi, GI = 2, OI = 5. IL-10 and IL-4, uninfected (0) = 6; 3, 9, 12 dpi, GI = 6, OI = 6; 17 dpi, GI = 3, OI = 8. Statistical analysis was performed using GraphPad Prism 5. *p = 0.05; **p = 0.01; ***p = 0.001. (C) Course of parasite distribution in oral infection. Male BALB/c mice were infected in the oral cavity (OI) with 1 × 10⁶ trypomastigotes forms of T. cruzi expressing luciferase (Dm28c-luc). Representative in vivo bioluminescence images were acquired in the same mice (n = 6), at 7 and 21 dpi, after 15 min of d-luciferin intraperitoneal administration (150 mg/kg), using IVIS^® Lumina image system (Xenogen). (D) T. cruzi loads in orally infected mice. Male BALB/c mice were infected in the oral cavity (OI) with 1 × 10⁶ trypomastigotes forms of T. cruzi expressing luciferase (Dm28c-luc). Organs and tissues were harvested for qPCR analysis to determine the parasite load (parasite equivalent/g) at 60 min, 7, and 21 dpi. The qPCR was performed in multiplex, targeting T. cruzi nuclear satellite DNA (Sat DNA) and IAC (internal amplification control), as a quality control. Parasite load in the nasal cavity (n: 60 min and 7 dpi = 5; 21 dpi = 4), esophagus (n: 60 min = 4; 21 dpi = 3), stomach (n: 60 min and 7 dpi = 4; 21 dpi = 3), small intestine (n: 60 min = 5; 7 dpi = 3; 21 dpi = 4), large intestine (n: 60 min = 5; 7 and 21 dpi = 4), and mandibular lymph nodes (n: 60 min = 4; 7 and 21 dpi = 3). Red dots: no parasite detection. Values present mean ± SEM. Kruskal–Wallis (Dunn’s post-test) for group kinetics. Statistical analysis was performed using Graph Pad Prism 5. *p < 0.05, **p < 0.01. Adapted from Barreto-de-Albuquerque et al. (51) and Silva-dos Santos et al. (52).