Yingyu Zhang ¹ Hiba Rehman ² Farina Khattak ³ Bushra Nisar Khan ⁴ Sadia Chaman ⁵ Ayaesha Riaz ⁶ Muhammad Ovais Omer ⁷ Aqib Ali ² Qamar Un Nisa ² Muhammad Mudassir ⁸ Gulbeena Saleem ^2*

• ¹ The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan Province, China
• ² Department of Pathology, University of Veterinary and Animal Sciences, Lahore, Pakistan
• ³ Scotland's Rural College, Edinburgh, Scotland, United Kingdom
• ⁴ Department of Zoology, University of the Punjab, Lahore, Punjab, Pakistan
• ⁵ Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences, Lahore,, Pakistan
• ⁶ Faculty of Veterinary and Animal Science, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Punjab, Pakistan
• ⁷ Department of Pharmacology and Toxicology, University of Veterinary and Animal Sciences, Lahore, Pakistan
• ⁸ Institute of Biochemistry and Biotechnology, University of Veterinary Animal Sciences, Lahore, Pakistan

Background: Amid growing concern about antimicrobial resistance due to the irrational use of antibiotics in treating common poultry diseases, particularly Salmonella which is a foodborne pathogen in humans. This study investigates the effects of ethnoveterinary supplementation of Rauwolfia serpentina (L. Benth. ex Kurz) powder (RSP) on three key immune-related genes; Suppressor of cytokine signaling 3 (SOCS3), the quiescence-related gene P20K (P20K), and the major histocompatibility complex Class IIβ (MHC class IIβ), gut morphology and growth performance of broiler chicks infected with Salmonella Gallinarum .Methods: Two hundred and forty day-old Hubbard classic chickens were randomly assigned to four groups: non-challenged control (NC), and Salmonella Gallinarum challenge group (SGC), and two treatment groups fed a basic diet supplemented with 1.5% Rauwolfia serpentina powder (RSP) with SGC (RSP-1) and 3% RSP with SGC (RSP-2), respectively, from day 3 till 28 days of age. Each treatment was replicated 4 times with 15 bird/replicate pen. On day 7, all the birds in the RSP-1, RSP-2 and SGC groups received 1 ml of BHI broth containing 2×10⁸ CFU of Salmonella Gallinarum via oral gavage. While control birds received an equivalent volume of sterile BHI broth. Gene expression analysis was conducted using real-time PCR to measure the expression of key immune-related genes: SOCS3, P20K, and MHC Class IIβ in spleen, liver, and caeca. Additionally, histopathological assessments of gut and growth performance parameters including feed intake, body weight gain, and feed conversion ratio (FCR) were monitored throughout the experimental period.The gene expression analysis at 3 and 21 days post-challenge revealed that SGC birds had significantly higher SOCS3, P20K, and lower MHC class IIβ expression (P <0.001) in the caecum, liver, and spleen of broiler chickens. In contrast, the RSP-1 and RSP-2 groups showed significantly lower SOCS3 and P20K expression (P < 0.001), alongside improved gut morphology, weight gain, and FCR compared to the SGC group, with these benefits increasing over time.In conclusion, these findings suggest that Rauwolfia serpentina supplementation modulates key immune-related gene expression (SOCS3, P20K, and MHC class IIβ), enhances intestinal health, and improves growth performance in broilers challenged with Salmonella Gallinarum.