Horse immune response of recombinant Lawsonia intracellularis vaccine: Assessing the immunomodulatory impact of probiotic supplementation.

Equine proliferative enteropathy (EPE), caused by Lawsonia intracellularis induces intestinal epithelial hyperplasia, resulting in malabsorption, weight loss, colic, and diarrhea. Despite its clinical and economic importance, no equine-specific vaccine is commercially available. Recombinant subunit vaccines are a safe and scalable alternative; however, their immunogenicity often requires improvement. Probiotic supplementation with Bacillus toyonensis may act as an immunomodulatory strategy to potentiate vaccine efficacy. This study aimed to evaluate the immunogenicity of a recombinant protein fragment derived from the li0902 gene of L. intracellularis, fused to the tetanus toxin (TT-Th) molecule, in horses, and to determine the impact of B. toyonensis supplementation on vaccine-induced humoral and cellular immune responses. The rLiTT antigen was expressed in Escherichia coli BL21 (DE3) and characterized by SDS-PAGE and Western blotting. Horses were vaccinated with rLiTT, with or without B. toyonensis supplementation. Serum IgG and IgG subisotypes were quantified by ELISA, and cytokine gene transcription in peripheral blood mononuclear cells was analyzed by qPCR. Vaccination with rLiTT elicited a specific humoral response in all horses. Supplementation with B. toyonensis significantly enhanced total IgG, particularly IgGb, and modulated IgGa and IgGT responses. Cytokine analysis showed an upregulation of mRNA transcription for il6 (∼9-fold), il4, tnf-α (∼5-fold), and il10, which was undetectable in unsupplemented animals. Conversely, il12 and il1 transcription decreased, suggesting attenuation of early pro-inflammatory signaling. The rLiTT protein represents a promising vaccine candidate against L. intracellularis. B. toyonensis supplementation enhanced both humoral and cellular immunity, supporting its potential as a probiotic adjuvant in equine vaccination strategies.