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

SIMPLE SUMMARY: Pullorum disease (PD), caused by Salmonella enterica serovar Pullorum (S. Pullorum), is the most consequential poultry disease in countries with a developing poultry industry, which poses a considerable economic burden on the poultry industry. Antimicrobial peptides are key components of the innate immune system and are next-generation antibiotic agents with broad antimicrobial spectrum, low resistance, and low cytotoxicity. The aim of the present study was to generate a recombinant antimicrobial peptide, OaBac5mini, and explore its efficacy on PD. In this study, 1-day-old chicks were orally challenged with S. Pullorum to establish PD models. S. Pullorum markedly increased the organ indexes of the heart, liver, spleen, and kidney; induced histopathological changes in multiple organs; and impaired the innate immunity through the TLR4/MyD88/NF-κB pathway. Recombinant OaBac5mini was generated by an Escherichia coli recombinant expression system and exhibited strong antibacterial activity in S. Pullorum-challenged chicks. It decreased the organ bacterial loads in the liver and spleen, ameliorated the organ indexes and histopathological changes of chicks with PD, and reduced the expression of pro-inflammatory cytokines by modulating the innate immunity through the TLR4/MyD88/NF-κB pathway. These findings reveal the in vivo antibacterial activity of recombinant OaBac5mini against S. Pullorum and demonstrate its therapeutic potential as an antibiotic agent for PD. 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.