Efficacy and safety evaluation of artificial intelligence-identified antimicrobial peptides for use against avian pathogenic Escherichia coli in the poultry industry

The overuse of antibiotics in both veterinary and human medicine has resulted in the emergence of antibiotic-resistant bacteria, prompting a search for effective alternatives. Antimicrobial peptides (AMP) are short, often cationic, peptide-based molecules with antimicrobial and immunomodulatory activity, which makes them promising alternatives to conventional antibiotics in poultry production. From a prior machine-learning-guided screen of 875 candidate AMP, 62 exhibited activity against avian pathogenic Escherichia coli (APEC) and low in vitro hemolytic and cytotoxic activity. We selected three lead AMP from this list (named TeRu4, TeBi1, and PeNi4), and evaluated their in vitro and in vivo efficacy, safety, and immunomodulatory potential for use in poultry farming. In animal experiments, AMP were administered via in ovo injection on day 18 of embryonic development. In APEC challenge trials, yolk sacs were inoculated with APEC post-hatch to assess early chick mortality, while in pen trials, birds were raised in a commercial production setting for 35 days. For challenged birds, TeBi1 (10 μg/egg) significantly reduced bacterial detection in the air sac and pericardium, increased body weight by 50% and reduced cytokine transcript levels by 10-30% on day 7 post hatch. In HD11 chicken macrophage-like cultured cells, TeRu4 (16 μg/mL) suppressed lipopolysaccharide (LPS)-induced pro-inflammatory cytokine transcript levels. In pen trials, TeRu4 (20 μg/egg) increased the survival probability of female birds by 4.9%, while TeBi1 (20 μg/egg) increased the survival probability of all birds by 4.4%, by day 35. Gene expression analysis revealed AMP- and sex-specific cytokine responses. In pen trials, no significant differences were observed in mean weights, feed conversion ratio (FCR), and flock uniformity on day 35. These findings demonstrate that the three selected AMP are safe antibiotic alternatives that improve survival, modulate immune responses, and maintain normal growth performance in broiler chickens.