Recombinant Antimicrobial Peptide OaBac5mini Alleviates Inflammation in Pullorum Disease Chicks by Modulating TLR4/MyD88/NF-κB Pathway

Abstract

Pullorum disease (PD), caused by Salmonella Pullorum (S. Pullorum), is a serious threat to the poultry industry worldwide. Antimicrobial peptides (AMPs) have drawn extensive attention as new-generation antibiotics because of their broad antimicrobial spectrum, low resistance, and low cytotoxicity. AMP OaBac5mini exhibits strong antibacterial activity against Gram-negative bacteria, but its efficacy and anti-inflammatory effects on chicks with PD remain unclear. The aim of this study was to generate recombinant OaBac5mini via the Escherichia coli (E. coli) recombinant expression system and evaluate its antibacterial effect against S. Pullorum in vitro and in vivo. Real-time cellular analysis (RTCA) results showed that recombinant OaBac5mini exhibited no cytotoxicity on IPEC-J2 and RAW 264.7 cells and significantly alleviated the drop in the cell index of S. Pullorum-infected cells (p < 0.0001). In the chick model of PD, recombinant OaBac5mini significantly attenuated the increase in organ indexes (heart, liver, spleen, and kidney) and bacterial loads (liver and spleen) induced by S. Pullorum. Histopathology examination showed that recombinant OaBac5mini ameliorated histopathological changes and inflammation in chicks with PD, including impaired epithelium of duodenal villi, infiltration of pseudoacidophilic granulocytes in the cecum and bursa of Fabricius, congested blood clots and increased macrophages in the liver, and increased lymphoid nodule and B lymphocytes in the spleen. Western blot and quantitative real-time PCR (qRT-PCR) results indicated that recombinant OaBac5mini alleviated inflammation by modulating innate immunity through the TLR4/MyD88/NF-κB pathway and by suppressing the expression of pro-inflammatory cytokines. These results suggested that recombinant OaBac5mini has good potential as a clinical substitute for antibiotics in PD intervention.