**Background:** Chagas disease (CD), caused by Trypanosoma cruzi, affects approximately 7 million people in Latin America and is a leading cause of heart failure. Current treatments—nifurtimox and benznidazole (BNZ)—have been used for over 40 years but are limited by side effects and low cure efficacy (50–80% in acute phase, 8–20% in chronic phase). Additionally, naturally resistant T. cruzi strains exist. There is an urgent need for novel therapeutic options. Nitazoxanide (NTZ), a broad-spectrum antiparasitic thiazolide, and electrolyzed oxidizing water (EOW), a disinfectant with potential immunomodulatory properties, have been proposed as candidate treatments. The canine model closely mimics human CD pathogenesis, making it suitable for evaluating new therapies.
**Methods:** Sixteen Náhuatl dogs (2–11 years, 12 males, 2 females, 14–27 kg) were confirmed negative for anti-T. cruzi antibodies by ELISA before the study. Dogs were randomly divided into six groups: non-infected untreated controls (HEALTHY, n=2), T. cruzi-infected NTZ-treated (Tc/NTZ, n=4), T. cruzi-infected EOW-treated (Tc/EOW, n=4), T. cruzi-infected BNZ-treated (Tc/BNZ, n=2), T. cruzi-infected untreated (Tc/UNT, n=2), and acute T. cruzi-infected untreated (TcA/UNT, n=2). Infection was established via intraperitoneal injection of 1.25 × 10^6 blood trypomastigotes of the Mexican H8 strain (DTU I). Treatments began at 20 days post-infection (dpi) and were administered orally for 10 consecutive days: NTZ at 50 mg/kg/day, EOW at 200 mL (60 ppm)/animal/day, and BNZ at 40 mg/kg/day. Anti-T. cruzi IgG antibodies were measured by ELISA at pre-infection, 1.5, 3.5, 9.5, and 12 months post-infection (mpi). Cytokines (IL-1β, IL-6, IL-10, IL-12B, IFN-γ, TNF-α) were measured at pre-infection, 47 dpi, and 15 days post-treatment (dpt). Electrocardiograms (EKG) were recorded pre-infection and at 3, 6, 9, and 12 mpi. Euthanasia was performed at 12 mpi (30 dpi for TcA/UNT). Hearts were examined macroscopically and histologically. Organ indices (heart, spleen, popliteal lymph node weight/body weight × 100) were calculated. Statistical analysis used Kruskal–Wallis test with significance at p < 0.05.
**Key Results:** Parasitemia was not detected by light microscopy in any infected group, but infection was confirmed by IgG seropositivity at 47 dpi in all infected groups. At 1.5, 3.5, and 9.5 mpi, Tc/NTZ, Tc/EOW, and Tc/UNT groups remained seropositive. The Tc/BNZ group became seronegative from 9.5 mpi. At 12 mpi (11 months post-treatment), both Tc/NTZ and Tc/EOW groups showed negative seroconversion, while Tc/UNT remained positive. Cytokine analysis showed that the Tc/NTZ group had increased proinflammatory cytokines (IL-6, TNF-α, IFN-γ, IL-12B, IL-1β) with moderate IL-10. The Tc/EOW group showed predominance of IFN-γ and IL-12B, with significantly high IL-1β (p = 0.0152) and IL-10 (p = 0.0247). The Tc/BNZ group showed an IL-1β increase without predominance of other proinflammatory cytokines. EKG findings showed alterations from 3 mpi in all infected groups. The Tc/NTZ group showed ventricular premature complexes, intraventricular conduction defects, low QRS voltage, right atrial enlargement, and right bundle branch block in one individual at 12 mpi. The Tc/EOW group showed persistent alterations with increased severity at 12 mpi in 50–75% of individuals, including right atrial enlargement, left ventricular enlargement, low QRS voltage, and bundle branch blocks. Macroscopic cardiac examination at 12 mpi revealed rounded cardiac silhouette, pale epicardium, ventricular dilatation, valvular endocardiosis, and pericardial fluid accumulation in the Tc/UNT group. These findings were present in 25–75% of Tc/NTZ and 25–100% of Tc/EOW groups. Heart index did not show statistically significant cardiomegaly in any group, though one dog each from Tc/UNT (50%), Tc/NTZ (25%), and Tc/EOW (25%) developed cardiomegaly. Splenic index showed statistically significant splenomegaly (p = 0.034) in all infected groups versus HEALTHY. Histopathology revealed lymphoplasmacytic interstitial myocarditis in all infected groups. Tc/NTZ, Tc/EOW, and Tc/BNZ groups showed mild inflammation; Tc/UNT showed severe coalescent inflammation. No fibrosis, edema, or amastigote nests were observed in chronic-stage groups. The TcA/UNT group showed moderate multifocal inflammation with intracellular amastigote nests.
**Clinical Implications:** This study provides the first evidence that NTZ and EOW, administered orally for 10 days, can induce seronegativity and reduce cardiac inflammation severity in a canine model of chronic Chagas disease, with efficacy comparable to benznidazole. NTZ induced a more favorable proinflammatory Th1 immune response than EOW. Neither treatment prevented electrocardiographic conduction abnormalities, but both reduced the magnitude of cardiac lesions. These findings are particularly significant because no veterinary drugs are currently approved for Chagas disease in dogs. NTZ and EOW could represent accessible, low-toxicity therapeutic options for pets and working dogs in endemic regions. However, the study's small sample size (n=2–4 per group) and lack of statistical significance for many outcomes limit the strength of conclusions. Further research with larger sample sizes, combination therapies, and longer follow-up is needed before clinical translation.