Probiotics as Antiparasitic Agents: Anti-Cryptosporidium efficacy of Lactobacillus Species, Bacillus proteolyticus, and Cell-Free Supernatants in Experimentally Infected Mice

Background Cryptosporidium parvum is a major zoonotic parasite causing high mortality in young ruminants and significant economic losses in livestock production. Current pharmacological interventions have limited efficacy, increasing the need for novel alternative therapies. This study evaluated probiotics and their cell-free supernatants (CFS) as alternative therapies. Methods This study identified two Lactobacillus subspecies ( strain 1, strain 2 ) and evaluated with Bacillus proteolyticus for their bacteriocin, and enzyme production. Based on bacteriocin and enzymes production, three formulations were prepared: single culture ( strain 1 , G1), mixed culture ( strain 1, strain 2 and B. proteolyticus , G2), and (mixed CFS, G3). These preparations were evaluated for their therapeutic effects against zoonotic Cryptosporidium parvum infection in experimentally infected mice. The experiment followed Factorial Two Ways Design. The data obtained was subjected to analysis of variance (ANOVA). Duncans Multiple Range Test were used to compare between means of treatment at probability 5%. Results Two Lactobacillus subspecies (Strain 1, L. bulgaricus and Strain 2, L. fermentum ) were identified. According to the results of in vitro analyses for antibacterial and enzymatic activities, strain 1 exhibited the highest activity. Strain 1 was used as a single probiotic culture, G1 and in combination with strain 2 and Bacillus proteolyticus to form a mixed probiotic culture, G 2). Treatment with G3 (CFS) produced the largest reduction in oocyst shedding and the lowest histopathological lesion scores. Immunological analyses showed treatment groups had heightened Immunoglobulin G (IgG), Interleukin-15 ( IL-15), and Interferon-gamma (IFN-γ) levels. G3 led in IgG response, G2 had the highest IL-15, and robust IFN-γ responses were seen in G1, G2, and NTZ-treated mice. Conclusion Single-strain probiotics enhance targeted immune responses, mixed strains promote broader immunomodulation, and postbiotics (CFS) present potent antiparasitic and immunostimulatory effects. The results offer a safe and effective alternative strategy for managing C. parvum infections.