Animal Ethics Committee of the University of Veterinary and Animal Sciences, Lahore, approved all the experimental procedures (Ref. No. DR/350 dated 24-07-2023).

Rauwolfia serpentina root material was purchased from Dr. Masood Pharmaceuticals (Pvt.) Ltd., Lahore, and authenticated by a taxonomist at Government College University, Lahore (specimen voucher: GC.Herb.Bot.2324). The roots were washed, dried and milled into a powder that was used in the birds' diet. Two experimental diets were used one with 15 and 30 g/kg Rauwolfia serpentina root powder in the diet. A proximate analysis (18) and phytochemical analysis of Rauwolfia serpentina were performed for qualitative and quantitative screening of the plant.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis was performed using an Agilent Technologies GC-MS system (California, USA) with a 6850 Network GC system and 5973-mass selective detector. Compound identification was done by comparing with the NIST 05 Mass Spectral Library and published spectra (24).

A total of 240 Hubbard Classic chickens were randomly assigned to four experimental groups, each consisting of four replicates with 15 birds per replicate. The treatment groups included: non-challenge control (NC), Salmonella Gallinarum challenge only (SGC), 1.5% Rauwolfia serpentina powder + challenge (RSP-1), and 3% Rauwolfia serpentina powder + challenge (RSP-2). Bacteriological analysis of fecal samples and serum plate agglutination test using Salmonella Gallinarum antigen was performed to make sure Salmonella-free status of chickens (25). On day 7, all the birds in the SGC group, RSP-1 and RSP-2 received the Salmonella Gallinarum challenge. In addition, challenged birds in RSP-1 and RSP-2 groups received a diet supplemented with 1.5% (15 g/kg) and 3% (30 g/kg) of Rauwolfia serpentina root powder respectively from day 3 till day 28 of age. During the experiment, all birds were housed in floor pens within climate-controlled rooms. The temperature was maintained between 33 and 30°C during the first 14 days, after which it was gradually reduced to 21°C by day 28. Throughout the study, all birds had unrestricted access to feed and water.

Salmonella Gallinarum stock, previously used in Liaqat et al. (26), was thawed and cultured on Salmonella Shigella Agar (Oxoid, UK) and Brilliant Green Agar (Oxoid, UK). An individual bacterial colony was transferred into Brain Heart Infusion Broth (Oxoid, UK) and allowed to grow overnight in an incubator maintained at 37°C. Birds in the challenge groups received 1 ml of BHI broth containing 2 × 108 CFU of Salmonella Gallinarum via oral gavage on day 7, while control birds received an equivalent volume of sterile BHI broth. Following the challenge, chickens were monitored twice daily for signs and symptoms of fowl typhoid, as described by Saleem et al. (2) till the end of the experiment.

On days 3 and 21 post-challenge, five birds from each replicate were humanely euthanized by cervical dislocation for post-mortem examination. Following necropsy, tissue samples from the caeca, spleen and liver were aseptically collected to evaluate expression levels of SOCS3, P20K and MHC CLASSIIβ genes that represent classical immune response, and stored in RNAlater^® solution (Thermo Fisher Scientific, Waltham, MA, USA) at −80°C until further processing.

Total RNA was extracted using TRIzol^® Reagent (27) according to the manufacturer's recommendation. Quantity and quality of RNA were assessed using a NanoDrop™ 2000 spectrophotometer and Bioanalyzer (Agilent 2100). Samples with an RNA Integrity Number (RIN) above 8.0 were utilized for subsequent analyses.

For the synthesis of complementary DNA (cDNA), 1 μg of RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time PCR (qPCR) was conducted in technical triplicate with SYBR Green chemistry using a Thermal Cycler (Applied Biosystems™ Veriti). The reactions were carried out in a total volume of 20 μl, containing 10 μl of 2 × SYBR Green Master Mix (Thermo Fisher Scientific), 0.5 μl of each forward and reverse primer (10 μM), 2 μl of cDNA template, and 7 μl of nuclease-free water. GAPDH was used as the endogenous control. The thermal cycling conditions included an initial denaturation at 95°C for 10 min, followed by 45 cycles of a three-step touchdown protocol (15). Melt curve analysis was performed to confirm amplification specificity. Gene expression levels for SOCS3, P20K, and MHC CLASSIIβ were quantified using previously validated primers (15). Primer sequences and gene accession numbers are provided (in Supplementary Table 1). Relative gene expression was determined using the 2^−ΔΔCt formula (28).

Caecal tissue samples from five birds from each replicate were collected on day 21 post-infection (in addition to gene analysis), washed with sterile saline solution to remove the digesta immersed in fixative (10% buffered formalin) followed by embedding in paraffin and cutting into 4 μm sections (29) and stained with H&E and Periodic Acid–Schiff (PAS). The slides were analyzed using a light microscope (Olympus CX31) with a digital imaging system (Olympus DP20, Olympus USA). The morphometric analysis included measurements of villus height (VH), crypt depth (CD), VH and CD ratio, and surface area calculated using the formula (2π) (VW/2) (VL). Goblet cell numbers, acidic and neutral mucin were also assessed (30).

Body weights (BW) were recorded for each replicate at day 0, 7 and 28 days of age. Body weight gain was calculated using the initial BW at the beginning and on days 7 and 28 days of age. Feed intake was also recorded at day 7 and 28 days of age. FCR were calculated to assess the growth performance of birds.

Data were analyzed using Genstat 11 for Windows (VSN International Ltd, Hemel Hempstead, UK). For qPCR data, relative gene expression was calculated using the 2^−ΔΔCt method. The pen served as the experimental unit for growth performance data, while the individual bird was the unit for histopathological, morphometric, and gene expression analyses. Morphometric parameters, including villus height (VH), crypt depth (CD), VH:CD ratio, surface area, and goblet cell counts, as well as mucin composition (acidic and neutral), were analyzed using analysis of variance (ANOVA) to evaluate the effects of treatments on caecal histomorphometry and mucin production. No outliers were removed prior to analysis. Gene expression data were then analyzed using a one-way analysis of variance (ANOVA). Post-hoc comparisons were conducted using Bonferroni's test to determine significant differences between specific groups. Differences were considered statistically significant at p < 0.05. Results are presented as means ± standard error of the mean (SEM).

During daily clinical observations, ~15% of the birds in the SGC group exhibited symptoms consistent with fowl typhoid, including ruffled feathers, rapid respiration, emaciation, and yellowish-white diarrhea. During the experiment, 1% of the chickens died. None of these deaths showed evidence of being caused by the Salmonella Gallinarum infection. On day 10 (3 days post-challenge), a post-mortem examination revealed moderate liver discolouration with hemorrhages and caeca lesions, predominantly inflammation, along with splenomegaly in the SGC group (Figure 1). This indicated a moderate severity of the challenge. In contrast, birds supplemented with Rauwolfia serpentina at 1.5% and 3%, exhibited milder effects with only three out of 10 and two out of 10 birds, respectively, showing mild liver discolouration with necrotic foci. Additionally, only a very mild degree of enteritis was observed in these birds.

The expression levels of three key immune-related genes-SOCS3, P20K, and MHC class IIβ were analyzed in the caecum, liver, and spleen of broiler chicks at 3 and 21 days post-challenge against Salmonella Gallinarum. The effects of Rauwolfia serpentina supplementation were compared to both NC and SGC (Figure 2).

On day 3 post-challenge, SOCS3 gene expression varied significantly across treatments and tissue (caeca, liver, and spleen: p < 0.05). The SGC birds exhibited the highest SOCS3 expression levels in the caecum, liver, and spleen, while birds supplemented with RSP-1 and RSP-2 showed significantly lower expression levels than the SGC group (Figure 2A). In the spleen, SOCS3 expression in RSP-1 and RSP-2 groups was still significantly higher than in the NC group (p < 0.05).

Supplementation of Rauwolfia serpentina had a significant effect on SOCS3 gene expression in the liver and spleen (p < 0.001) on day 21 post-challenge, while no significant differences (p = 0.061). were observed in the caeca (Figure 2B). SGC birds exhibited the highest SOCS3 expression in both the liver and spleen, with significantly lower expression observed in birds supplemented with Rauwolfia serpentina (RSP-1 and RSP-2) compared to the SGC group. However, SOCS3 levels in the supplemented groups remained higher than in the unchallenged birds. Both supplementation levels (1.5 and 3%) resulted in similar SOCS3 expression in the spleen, significantly lower than the SGC group but higher than the unchallenged birds (Figure 2B).

On day 3 post-challenge, P20K gene expression showed tissue-specific variations across treatment groups (Figure 2A). In all three organs, both RSP-1 and RSP-2 groups exhibited significantly lower P20K expression than SGC groups (p < 0.05). Expression of P20K was highest in the SGC group, significantly higher than all other groups (p < 0.05). In the liver, supplementation of Rauwolfia serpentina resulted in intermediate expression levels, significantly higher than NC but lower than SGC (p < 0.05). In the spleen, P20K expression was significantly elevated in all SGC group compared to the other treatment group (p < 0.05). At 21 days post-challenge, caecal P20K expression showed no statistically significant differences among groups (p = 0.111, Figure 2B).

MHC class IIβ gene expression patterns varied across tissues and treatment groups on day 3 post challenge (Figure 2A). SGC groups showed significantly lowest expression in all three tissues examined compared to all other treatment groups (p < 0.05). Liver MHC class IIβ expression was significantly elevated in all treatment groups compared to the NC group (p < 0.05). The SGC group showed the lowest expression, followed by RSP-1 and RSP-2, all significantly different from the NC group (p < 0.05). On day 21 post-challenge, significant differences were observed in MHC Class IIβ expression across all tissues (caeca: p = 0.008, liver and spleen: p < 0.001). SGC birds exhibited the lowest expression levels in the caecum, liver, and spleen, whereas birds supplemented with RSP-2 and RSP-1 showed significantly higher expression levels than SGC group (Figure 2B).

Histopathological examination of caecal tissue revealed marked disruption of normal architecture and abnormal mucin production in SGC birds (Figure 3B). Alcian Blue staining showed intense mucin production, accompanied by areas of necrosis, tissue degeneration and inflammatory infiltrates (Figure 3F). These findings suggest a progressive pathological process affecting the tissue, likely impairing nutrient absorption and mucosal defense. In contrast, birds supplemented with RSP-2 and RSP-1 showed only light to moderate changes, with mucin production and tissue architecture largely intact. Cellular morphology appeared mostly regular, with only slight nuclear size and shape variations, suggesting that Rauwolfia serpentina supplementation contributed to tissue repair.

Morphometric analysis indicated that Rauwolfia serpentina supplementation had a significant impact on caecal morphology (Table 4). Villus height (VH) was significantly greater in the RSP-2 (707 μm) and RSP-1 (706 μm) groups compared to the SGC group (267 μm; p = 0.008). Crypt depth was also preserved in the RSP-2 (173 μm) and RSP-1 (167 μm) groups, significantly higher than in the SGC group (74.3 μm; p = 0.002). Although the VH ratio did not differ significantly among the groups (p = 0.675), the surface area of the caecum was significantly larger in the RSP-2 (738 μm) and RSP-1 (743 μm) groups compared to the SGC group (145 μm; p = 0.005). The number of goblet cells in the RSP-2 group was significantly higher (p = 0.01), with an average of 12 cells per section, compared to five cells in the SGC group. Acidic and neutral mucin levels did not differ significantly between treatments (p = 0.079 and p = 0.222, respectively).

During the first week of age, no significant differences in weight gain, FCR, or feed intake were observed among the treatment groups. However, by day 28 (21 days post-challenge), Rauwolfia serpentina supplementation had marked effects on growth performance (Table 5). Birds in the RSP-2 group showed significantly higher weight gain (1,687 g) compared to other groups received SGC (p = 0.006) except NC group. The SGC group showed lower overall weight gain (1,455 g) compared to all other treatment groups (p = 0.001). The RSP-2 group also had significantly lower feed intake (2,379 g) than the other groups received SGC (p = 0.003), indicating better feed efficiency. In contrast, feed intake in the RSP-1 group (2,456 g) was comparable to the control groups. These results suggest that Rauwolfia serpentina supplementation at both levels offered protective effects against SGC, with the effects becoming more pronounced over time.

Present study revealed significant variation in the expression of three key immune-related genes- SOCS3, P20K, and MHC class IIβ in broiler chicks supplemented with Rauwolfia serpentina and challenged with Salmonella Gallinarum. Supplementation with Rauwolfia serpentina, significantly modulated the tissue specific expression of these genes in response to Salmonella Gallinarum challenge.

In particular, gene expression patterns varied across the caecum, liver, and spleen. SOCS3 was significantly upregulated in the SGC group, followed by P20K, while MHC class IIβ expression was lowest in these tissues. The differential expression of immune-related genes in these organs following pathogen exposure underscores the importance of a coordinated immune response during the early stages of systemic infection (15). Following the invasion, Salmonella activates the host immune responses by invading macrophages and subsequent activation of pro-inflammatory cytokines such as IL-6 and TNF-α, responsible for the bird's innate and adaptive immune responses.

Suppressor of cytokine signaling 3 (SOCS3) plays a crucial role in regulating TNF-α, IL-6, and IL-12 (31, 32). In mouse dendritic cells, SOCS3 has been found to inhibit IL-12 expression, which can shift the immune response toward a Th2 profile along with decreased expression of MHC class IIβ (33). This explains the down-regulation of MHC class IIβ expression observed in SGC chicks. MHC class II, consisting of alpha and beta chains, is predominantly expressed on antigen-presenting cells such as macrophages, dendritic cells, and phagocytes. The variability of avian MHC haplotypes influences host susceptibility or resistance to various pathogens (34). In the current study, significant upregulation of SOCS3 in the caecum, liver, and spleen of birds challenged with Salmonella Gallinarum likely indicates an attempt to moderate the inflammatory response against infection (35). As the immune response of birds to Salmonella infection primarily involves a pro-inflammatory reaction, Salmonella is known to evade host immunity by upregulating the expression of SOCS3, thereby inhibiting the JAK/STAT pathway, leading to reduced production of cytokines like IFN-γ responsible for bacterial clearance and inhibition of MHC class IIβ expression to avoid detection of the immune response and persist in infection (36, 59).

A balanced adaptive immune response relies on the proper coordination and regulation of Th1 and Th2 cell activation, facilitated by Treg cells (37). The quiescence-related gene P20K (P20K) was significantly elevated in the liver and spleen of Rauwolfia serpentina-supplemented birds. This upregulation might indicate a Treg response, crucial in balancing the immune response to Salmonella Gallinarum. This differential expression of P20K across different tissues suggests that Rauwolfia serpentina might modulate host responses in a tissue-specific manner. This is particularly evident by an exhibition of different patterns of MHC Class IIβ expression across caeca, liver and spleen on days 3 and 21 post-challenge. MHC Class IIβ gene has a crucial role in initiating adaptive immune responses by presenting antigens to CD4+ T cells. On day 21 post-challenge, SGC birds exhibited the lowest expression of MHC class IIβ levels, whereas birds supplemented with Rauwolfia serpentina showed significantly higher expression levels indicating that Rauwolfia serpentina supplementation upregulated MHC class IIβ expression, in the spleen and caeca. This early immune response in current study is likely due to the initial activation of the immune system in response to both the dietary supplementation and Salmonella Gallinarum challenge. The downregulation of MHC class IIβ expression in the caecum of SGC birds aligns with previous findings (15), suggesting that Salmonella Gallinarum may evade host defense by suppressing MHC class IIβ expressions. Studies have shown that Salmonella Enteritidis can evade the Th1 immune response by inducing SOCS3 up-regulation, which in turn inhibits IFN-γ, leading to reduced MHC class IIβ expression (15, 38, 39). In contrast, the upregulated MHC class IIβ expression in the Rauwolfia serpentina-supplemented groups suggests that phytochemicals in the plant may promote persistent antigen presentation and enhance immune responses, even in the absence of active infection. This may be attributed to immune-boosting and anti-inflammatory properties of Rauwolfia serpentina (40). This tissue-specific difference observed in this study highlights the complexity of the immune responses to systemic pathogen challenges and aligns with previous studies of variable immune responses between systemic and localized immune systems in broiler chicks challenged with Salmonella Gallinarum (15). The immunomodulatory effects of Rauwolfia serpentina against Salmonella Gallinarum observed in the current study may be attributed to its potential chemical compounds, reserpine, saponins, tannins, flavonoids, and terpenoids. It particularly affects the release of norepinephrine with subsequent elevation of cytokine production and cell proliferation. We hypothesized that reserpine, along with other potential compounds present in Rauwolfia serpentina, may interplay host immune system, therefore activating several metabolic pathways. These pathways likely to enhance the production of antimicrobial peptides, enhanced IL-2 expression with reduced CTLA-4 expression therefore promoting T cell activation and proliferation in chickens (41). Additionally, reserpine has been shown to improve gut health by influencing antimicrobial pathways, mainly epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) and elevating AMP gene expression while decreasing the number of Enterobacteriaceae and Salmonella counts (10).

The results of the current study revealed mucosal damage, as indicated by a reduced VH and CD in broiler birds challenged with Salmonella Gallinarum. However, supplementation with Rauwolfia serpentina, especially in the RSP-2 group, resulted to an increase in goblet cell numbers in the caeca, suggesting improved gut health. Goblet cells are essential for mucus secretion, forming a protective barrier against pathogens and facilitating nutrient absorption (42). The current study further revealed a significant suppressive effect of the Salmonella challenge on goblet cell numbers, as indicated by a notable decrease in goblet cell numbers in the caeca of SGC broiler chickens. This finding aligns with observations of gut morphology, where a reduced VH to CD ratio was evident in broiler chicks that received Salmonella Gallinarum challenge. Prior research has similarly demonstrated a significant decrease in VH and CD in the intestines of Salmonella Typhimurium-infected birds, indicating potential mucosal damage caused by this pathogen.

Intestinal morphology, measured through duodenal and ileal VH and CD, as well as the VH to CD ratio, serves as an indicator of gut health in broilers (42). In addition to increase in goblet cells as evident by histomorphology (Figure 3D) coupled with improved villus morphology (Figure 3; Table 4), mainly increase in surface area that helps in better nutrient absorption, indicating a larger surface area for enzymatic activity and nutrient uptake (43) suggests that Rauwolfia serpentina root may enhance the intestinal barrier function and overall gut health in broiler chicks against Salmonella Gallinarum.

In the present study, growth performance parameters further support the potential benefits of Rauwolfia serpentina supplementation against Salmonella Gallinarum. During the first week of the trial, no significant differences were observed in feed intake, weight gain, or FCR across different treatment groups. However, by the fourth week, the RSP-2 birds demonstrated the best FCR (lower values) among all treatment groups, suggesting a more efficient conversion of feed into body mass. The enhanced growth performance in the RSP-2 group could be attributed to improved gut health, as evidenced by increased VH and goblet cell numbers, potentially leading to better nutrient absorption. Another possible explanation for better FCR could be attributed to a well-regulated immune response, as indicated by the upregulation of key immune-related genes, allowing birds to combat Salmonella Gallinarum challenge more effectively without excessively diverting energy from growth. The potential effects of Rauwolfia serpentina phytochemicals include promoting beneficial gut microbiota that contributes to improved nutrient utilization (41).

The observation that the birds in RSP-2 group showed slightly better performance in terms of better weight gain and FCR than RSP-1 could be due to the presence of additional beneficial bioactive compounds in the whole root powder or more release of active ingredients and more prolonged action of active compounds in the gut, promoting more sustained immune modulation and better growth performance. The finding that RSP supplementation results in better weight gain and FCR compared to Salmonella Gallinarum challenge may be due to multiple reasons, such as improved intestinal morphology leading to better nutrient absorption, reduced pathogen load, and lower inflammation, which allows more energy to be directed toward growth rather than immune responses.

Our findings suggest that Rauwolfia serpentina supplementation may offer a multifaceted approach to improving the health and productivity of broiler chicks challenged with Salmonella Gallinarum. The combination of enhanced immune gene expression, improved gut morphology, and better growth performance presents a promising picture for the use of this phytogenic as a natural growth promoter and immunomodulator.