Novel integrated approach modelling proanthocyanidins and bacteriophages to combat multi drug Salmonella Typhimurium in challenged broilers

Hanan Sulaiman Al-khalaifah¹Doaa Ibrahim^2*Ahmed Abdelfattah-Hassan³Dina Ibrahim⁴Alaaeldin Mohamed Saad⁵M. F.Saad⁵Reham. A. Elbhnsawy⁶Asmaa A. Azouz⁷Mayada Mahmoud⁸Sherief Mohamed Abdel-Raheem⁹Hesham Ismail⁹Rania M . S. El-Malt¹⁰Marwa Ibrahim Abd El-Hamid¹¹

• ¹Kuwait Institute for Scientific Research, Safat, Kuwait
• ²Department of Nutrition and Clinical Nutrition, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
• ³Department of Veterinary Medicine, College of Applied and Health Sciences, A’Sharqiyah University, P.O Box 42, Postal code 400, Ibra, Sultanate of Oman, Ibra, Oman
• ⁴Bacterioloy, Mycology, Immunology Department, Faculty of veterinary, medicine, Mansoura University, Mansoura, Egypt
• ⁵Zagazig University, Zagazig, Egypt
• ⁶Department of poultry and rabbit diseases, Veterinary teaching hospital, Faculty of veterinary medicine Mansoura university, Mansoura, Egypt
• ⁷Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt, Giza, Egypt
• ⁸Department of Microbiology, Faculty of Science, Ain Shams University, Cairo 11566, Egypt., Cairo, Egypt
• ⁹King Faisal University College of Veterinary Medicine, Al Ahsa, Saudi Arabia
• ¹⁰Department of Microbiology, Animal Health Research Institute (AHRI), Zagazig Branch, Agriculture Research Center (ARC), Zagazig 44516, Egypt, Zagazig, Egypt
• ¹¹Zagazig University Faculty of Veterinary Medicine, Zagazig, Egypt

The emergence of multidrug bacterial isolates, including Salmonella (S.) Typhimurium, which is primarily spread to humans through chicken products, is correlated with a rising prevalence of antimicrobial therapy failure. Thus, we performed a comprehensive analysis of the combined impact of Salmonella bacteriophage (BP) and grape seed oligomeric pro-anthocyanidins (GSOPs) on growth performance, immune functions, antioxidant capacity, cecal microbiota, gut integrity, and S. Typhimurium resistance in challenged broilers. A total of 250 Ross-308 male broiler chicks were offered either a control diet or a diet supplemented with Salmonella BP alone at concentrations of 109 PFU/ 0.1mL, GSOPs alone at a level of 400 mg/ kg diet, and a combination of both Salmonella BP and GSOPs and experimentally infected with MDR S. Typhimurium strain at 14 days of age. Broilers administered BP, GSOPs, and their combination, particularly BP+GSOPs, had enhanced growth performance attributes even following a challenge with S. Typhimurium, alongside decreased mortality percentage, which was evidenced by increased expression of MUC-2, β-defensin-1, cathelicidins-2, JAM-2, occludin, and CLDN-1 genes, reduced S. Typhimurium abundance, and downregulating its virulence-associated genes (sopE, and spvC), alongside restored intestinal histological features. GSOPs+BP fortified group exhibited higher cecal beneficial bacteria counts (Bacteroides, Firmicutes, Lactobacillus, and Bifidobacterium species), lower cecal harmful bacteria loads (Escherichia, Enterobacteriaceae, Clostridium clusters I, and IV), decreased serum oxidative markers (H2O2, ROS, and MDA), and increased serum antioxidant enzymes (SOD, CAT, and GSH-Px). The incorporation of dietary BP, and GSOPs combination significantly downregulated the intestinal inflammatory regulated genes (IL-1β, IL-6, CCL4, CCL20, COX-2, and iNOS), and mTOR gene, and reduced the serum concentrations of LYZ, NO, CRP, and complement C3, alongside increased serum IgG, and IgM levels, and upregulation of autophagy-related genes (atg5, atg7, atg12, BCLN-1, and LC3-II). The aforementioned beneficial effects of the BP and GSOPs combination support their prospective use in avian nutrition to enhance performance and stimulate immune defense against gastrointestinal illnesses, including S. Typhimurium infection.