Abstract

Host-derived probiotic strains offer an effective alternative to antibiotics for improving animal gastrointestinal health. In this study, two probiotic strains—Lactiplantibacillus plantarum TUCO-16 and Lacticaseibacillus rhamnosus TUCO-17—isolated initially from canine colostrum and milk, were scaled up and formulated with oligosaccharides into freeze-dried preparations. These formulations were evaluated in vitro using canine or human cell models to assess cytotoxicity, adhesion capacity, immune-related gene expression, reactive oxygen species (ROS) production, and paracellular permeability. The strains demonstrated high adhesion of up to 92% (p < 0.001), no cytotoxic effects, and induced significant (p < 0.0001) expression of both pro- and anti-inflammatory cytokines. They also stimulated the expression of mucins and tight junction proteins in epithelial cells up to increases of 414-fold and 21-fold (p < 0.0001), respectively. In addition, the formulations enhanced ROS production in macrophages (p < 0.0001) and maintained or reduced epithelial permeability (p < 0.0001). An in vivo trial in dogs with acute diarrhea confirmed the safety of the formulations. It showed improvement in stool consistency at the end of treatment without affecting frequency, contrasting with the effects of metronidazole. Overall, the results support using these host-specific probiotics as immunomodulatory agents that enhance intestinal barrier integrity and offer a safe, antibiotic-free alternative for managing acute diarrhea in dogs. © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2025.