Abstract
Background: Post-weaning diarrhea (PWD) in piglets, often caused by F4+ enterotoxigenic Escherichia coli (ETEC), poses significant challenges in pig production. Traditional solutions like antibiotics and zinc oxide face increasing restrictions due to growing concerns over antibiotic resistance and environmental sustainability. This study investigates the application of bivalent heavy chain variable domain (VHH) constructs (BL1.2 and BL2.2) targeting ETEC virulence factors, administered in feed to mitigate ETEC-induced PWD in weaned piglets. Results: The supplementation of BL1.2 and BL2.2 in both mash and pelleted feed significantly reduced the diarrhea incidence and fecal shedding of F4+ ETEC in challenged piglets. Pelleted feed containing VHH constructs helped to preserve gut barrier integrity by maintaining levels of the tight junction protein occludin in the small intestine. Additionally, the constructs maintained blood granulocyte counts at a similar level to the non-challenged control group, including neutrophils, and ameliorated the acute phase protein response after challenge. Notably, even at low feed intake immediately after weaning, VHH constructs helped maintain piglet health by mitigating ETEC-induced inflammation and the resulting diarrhea. Conclusions: Our findings demonstrated that using VHH constructs as feed additives could serve as an effective strategy to help manage ETEC-associated PWD, by reducing F4+ ETEC gut colonization and supporting gut barrier function of weaned piglets. The high stability of these VHH constructs supports their incorporation into industrial feed manufacturing processes, offering a more sustainable preventive strategy compared to traditional antimicrobial interventions, which could contribute to sustainable farming practices.