Isolation and identification of host-associated lactic acid bacteria with probiotic potential from Red Junglefowl (Gallus gallus) and preliminary in vivo evaluation in broiler chickens.

The gastrointestinal tract of host-associated birds may provide avian-adapted probiotic candidates for poultry production. This study isolated and characterized lactic acid bacteria (LAB) from Red Junglefowl (RJF; Gallus gallus), screened representative strains for probiotic-related in vitro traits, and performed a preliminary in vivo evaluation in broiler chickens. Thirty-nine presumptive LAB were recovered from the crop, small intestine, large intestine, and cecum of RJF birds and were assigned by near full-length 16S rRNA gene sequencing to Limosilactobacillus reuteri (28/39, 71.8%), Ligilactobacillus salivarius (8/39, 20.5%), and Weissella paramesenteroides (3/39, 7.7%), represented 14 sequence types. Representative strains were assessed for auto-aggregation, surface hydrophobicity, antagonistic activity against Escherichia coli and Salmonella sp., acid and bile tolerance, hemolysis, DNase activity, and phenotypic antibiotic susceptibility. Marked strain-to-strain variation was observed, but L. reuteri-1, L. salivarius-3, and W. paramesenteroides-3 showed the most balanced overall profiles and were selected for a multi-strain formulation. In a 35-d broiler trial, 108 Arbor Acres chicks were divided into three groups: a PBS control group, an RJF-derived candidate probiotic mixture (RJF-Mix) group, and a commercial probiotic product (CPP) group, with 6 pens per treatment and 6 birds per pen. All chicks had almost similar initial body weights, and there was no significant difference among treatments in total feed intake. However, final body weight was 2485.5 ± 37.3 g/bird in the RJF-Mix group and 2513.7 ± 23.4 g/bird in the CPP group, compared with 2288.6 ± 27.8 g/bird in the control group (P < 0.0001). The RJF-Mix (1.377 ± 0.006) and CPP group (1.364 ± 0.010) showed lower final cumulative feed conversion ratio as compared to the control group (1.517 ± 0.014; P < 0.0001). To our knowledge, this is the first study to recover host-associated LAB from the wild ancestor of the domestic chicken, RJF, and to advance selected isolates from a transparent, criterion-based in vitro prioritization to preliminary in vivo broiler validation against a CFU-matched commercial probiotic comparator. In conclusion, these findings indicate that RJF might be a promising natural resource of host-associated LAB with potential for poultry probiotic development; however, the three selected isolates are presented as promising probiotic candidates rather than fully validated probiotics, and further genomic, safety, and larger-scale validation are required.