Kinetics of Biomarkers for Therapeutic Assessment in Swiss Mice Infected with a Virulent Trypanosoma cruzi Strain

Abstract

Chagas disease (CD), caused by Trypanosoma cruzi, is a neglected tropical illness affecting 6-8 million people in Latin America. Reaching scholarly consensus on the host response to T. cruzi infection remains a significant challenge, primarily due to substantial heterogeneity in outcomes driven by both the choice of animal model and the infecting parasite's discrete typing unit (DTU). This variability complicates the evaluation and comparison of new therapeutic compounds against existing drugs, namely benznidazole and nifurtimox. This study provides a comprehensive, kinetic, multifaceted characterization of the acute infection using the highly virulent T. cruzi Y strain (TcII) in outbred Swiss mice. Here, crucial infection parameters are presented, including the optimal infective dose, the parasitemia dynamics, tissue damage markers, hematological profiles, cytokine production (Th1/Th2/Th17/Th22), and molecular parasite identification in target organs (heart, colon, esophagus, spleen, and liver) across the span of the infection. The novelty of this study lies in the kinetic integration of these parameters within a defined model; rather than presenting isolated data points, we demonstrate how the biochemical, physiological, and clinical signs and immunological responses, with the resulting organ involvement, evolve and interact over time. To complete the report, a necropsy evaluation was performed at the end of the acute, fatal infection, and it is presented here. This study fulfills a long-standing recommendation from diverse drug discovery groups for the creation of a definitive reference model to standardize preclinical testing for anti-Chagasic agents.

Keywords: Swiss mice; T. cruzi; animal model; chagas disease.

Figure 1

Parasitemia levels in Swiss mice infected with T. cruzi strain Y. Parasitemia was evaluated for 17 days. The number of parasites per milliliter (log₁₀ scale) was expressed in terms of mean and standard deviation. d.p.i.: days post-infection. *, ***, and **** represent significant differences at p < 0.005, p < 0.001, and p < 0.0001, respectively. ANOVA was performed, followed by Bonferroni’s test (two-tailed). The data correspond to two independent experiments. Different shapes represent different dates.

Figure 2

Clinical manifestations of acute infection in Swiss mice infected with T. cruzi strain Y. (a) An uninfected mouse and (b) a T. cruzi strain Y-infected mouse at 12 d.p.i., exhibiting hunched posture and piloerection. d.p.i.: days post-infection.

Figure 3

Kinetics of body temperature and body weight over time post-infection with T. cruzi. (A). Body temperature (°C) and (B). Body weight (g) of control (open circle) and T. cruzi strain Y-infected (black circle) mice were expressed as mean ± standard deviation. d.p.i.: days post-infection. * p < 0.05. Data were analyzed via ANOVA, followed by the Bonferroni test (two-tailed); n = 10 mice/time point.

Figure 4

Serum creatine phosphokinase (CPK), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels over time post-infection with T. cruzi strain Y. (A) CPK, (B) ALT, and (C) LDH serum levels in T. cruzi Y-infected mice were expressed as fold-change over uninfected animals ± standard deviation. d.p.i.: days post-infection. ** p < 0.01; **** p< 0.0001. Data were analyzed via ANOVA, followed by Bonferroni’s two-tailed test. The numbers above each bar indicate the number of animals per group. The data presented here take into account biochemical markers from experiments I and II.

Figure 5

Kinetics of total and differential white blood cell counts in control and T. cruzi strain Y-infected mice. (A). Total white blood cell count (WBC) expressed as fold increase over control mice at each time point (black bars). Absolute (right) and relative (left) counts of (B) monocytes, (C) granulocytes, and (D) lymphocytes from control (white bars) and T. cruzi strain Y-infected mice (black bars). Values are expressed as mean ± standard deviation. d.p.i.: days post-infection. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Data were analyzed through ANOVA, followed by Bonferroni’s two-tailed test. The numbers above each bar indicate the number of animals per group.

Figure 6

Kinetics of erythrocyte parameters in control and T. cruzi Y-infected mice. (A) total red blood cells (×10⁶/mm³), (B) hemoglobin (g/dL), (C) hematocrit (%), (D) mean corpuscular hemoglobin concentration (MCHC, g/dL), (E) mean corpuscular hemoglobin (MCH, pg), and (F) red cell distribution width (RDW, %) in peripheral blood in control (white bars) and T. cruzi strain Y-infected Swiss mice (black bars) were expressed as mean ± standard deviation and analyzed through ANOVA, followed by Bonferroni’s two-tailed test. The numbers above each bar indicate the number of animals per group. d.p.i.: days post-infection.

Figure 7

Platelet parameters. (A) platelet count (10³/μL), (B) plateletcrit (PCT, %), and (C) mean platelet volume (MPV, fL) in peripheral blood of control (white bars) and T. cruzi strain Y-infected Swiss mice (black bars). Values are expressed as mean ± standard deviation. d.p.i.: days post-infection. * p < 0.05, ** p < 0.01, and *** p < 0.001, ANOVA, followed by a two-tailed Bonferroni test, was used to analyze significance. The numbers above each bar indicate the number of animals per group.

Figure 8

Serum cytokine profiles of control and T. cruzi strain Y-infected Swiss mice. ELISA was used to measure serum levels of IL-17 and IL-22, while serum levels of TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-13 were assessed via flow cytometry. Only the cytokines of control (white bars) and T. cruzi strain Y-infected Swiss mice (black bars) showing statistically significant differences are presented: (A) IL-6, (B) IFN-γ, and (C) IL-22. * p < 0.05, ** p < 0.001, **** p < 0.0001. Two-tailed ANOVA, followed by Bonferroni’s test, was performed. The dotted line indicates the threshold.

Figure 9

Macroscopic comparison of solid tissues of control and infected mice on day 14 p.i. (A) control mouse; (B) infected mouse. The mouse drawing in the lower left corner of Figure A indicates which part of the mouse is open in the picture. Arrows point to organs that show morphological or colorimetric characteristics that differ from those in uninfected mice.

Figure 10

Comparative organ weights in infected and control mice. (A) Heart, (B) esophagus, (C) liver, and (D) spleen expressed as percentage of body weight in control (open circles) and T. cruzi strain Y-infected mice (black circles). Values are expressed as mean ± standard deviation. Data were analyzed using ANOVA followed by Bonferroni’s two-tailed test. * p < 0.05, ** p < 0.001, and **** p< 0.0001. d.p.i.: days post-infection.