Augmenting avian immunity naturally: A comprehensive evaluation of the immunomodulatory efficacy of Tephrosia purpurea extract in broiler chickens

Introduction

The global poultry industry is undergoing a paradigm shift toward sustainable and antibioticfree production systems due to growing concerns regarding antimicrobial resistance, drug residues in meat, and regulatory restrictions on antibiotic growth promoters. According to the World Health Organization (2023), antimicrobial resistance remains one of the top ten global public health threats, prompting urgent calls for alternative strategies to maintain animal health and productivity [1] . In this context, phytogenic feed additives and plant-derived immunomodulators have emerged as viable and eco-friendly alternatives to conventional chemotherapeutic agents. Immunomodulation refers to the alteration of immune responses through stimulation or suppression of specific and non-specific mechanisms. Herbal immunomodulators often exert their effects by enhancing macrophage activity, lymphocyte proliferation, cytokine production, and antibody synthesis [2,3] . Unlike synthetic immunostimulants, medicinal plants typically exhibit multi-targeted actions due to their diverse phytochemical constituents, including flavonoids, alkaloids, terpenoids, saponins, and phenolic compounds. Recent studies in poultry science have demonstrated that botanical extracts can improve vaccine responsiveness, enhance resistance to infectious diseases, and optimize growth performance under stress conditions [4,5] . Several medicinal plants such as Curcuma longa, Withania somnifera, Ocimum sanctum, Panax ginseng, and Zingiber officinale have been extensively studied for their immunomodulatory and adaptogenic properties in both laboratory animals and poultry species. These botanicals have demonstrated enhancement of antibody titers, stimulation of delayed-type hypersensitivity reactions, and improvement in lymphoid organ development.

Tephrosia purpurea (Family: Fabaceae), commonly known as Wild Indigo, is a perennial herb widely distributed across the Indian subcontinent. Traditionally used in Ayurvedic and Unani systems of medicine, the plant is reputed for its hepatoprotective, anti-inflammatory, antioxidant, antidiabetic, and anticancer properties [6] . Phytochemical investigations reveal that T. purpurea is rich in flavonoids, rotenoids, glycosides, sterols, and phenolic compounds, which are known to modulate immune signalling pathways and oxidative stress mechanisms [7] . Oxidative stress and immunosuppression are common challenges in intensive broiler production systems. Heat stress, vaccination, high stocking density, and pathogen exposure compromise immune efficiency and predispose birds to disease outbreaks. Emerging evidence suggests that plant-derived antioxidants can attenuate oxidative damage, thereby enhancing immune competence and overall performance [8] . Despite the documented pharmacological benefits of Tephrosia purpurea, limited scientific data are available regarding its immunomodulatory efficacy in poultry. Therefore, the present study was designed to systematically evaluate the immunomodulatory potential of methanolic extract of Tephrosia purpurea in broiler chickens by assessing both cell-mediated and humoral immune responses, haematological indices, and immune organ development under controlled experimental conditions.

Materials and Methods

Plant Material Collection, Authentication and Extraction

The whole plant of Tephrosia purpurea (Family: Fabaceae) was collected from a local agro-climatic region and authenticated by a qualified taxonomist. The plant material was shade-dried at ambient temperature (25-30 °C) to preserve thermo-labile bioactive compounds and subsequently pulverized into coarse powder using a mechanical grinder. Methanolic extraction was performed using Soxhlet apparatus following continuous hot percolation technique, a standardized method widely employed for efficient recovery of flavonoids and rotenoids [7] . Methanol was selected as the solvent due to its high polarity index and proven efficiency in extracting phenolic and immunoactive phytoconstituents [8] . The extract was concentrated under reduced pressure using a rotary evaporator and stored at 4 °C in airtight containers until further use to prevent oxidative degradation.

Experimental Birds, Housing and Ethical Compliance

All experimental procedures were carried out in accordance with guidelines prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India [9] . Institutional Animal Ethics Committee (IAEC) approval was obtained prior to the commencement of the study. A total of 36 day-old Vencobb broiler chicks were procured from a commercial hatchery and randomly allocated into three treatment groups with three replicates per group (Four birds per replicate). Randomization was performed to minimize selection bias [10] . Birds were individually identified using numbered leg bands. The birds were reared under standard deep litter system with controlled temperature (32 °C during first week with gradual reduction), relative humidity (60-70%), and 23-hour light schedule during starter phase as per commercial broiler management guidelines [11] . Feed and water were provided ad libitum throughout the experimental period of six weeks.

Experimental Design and Treatment Regimen

The study followed a completely randomized design (CRD). The treatment groups were as follows:  Control Group (T1): Basal diet  Standard Group (T2): Basal diet supplemented with Lincomycin @ 0.05% w/w  Test Group (T3): Basal diet + T. purpurea extract @ 0.04% w/v in drinking water

The basal diet was formulated to meet or exceed nutrient requirements for broiler chickens as recommended by the National Research Council [12,13] . Lincomycin was used as a standard antibiotic growth promoter owing to its established antimicrobial and growth-enhancing properties in poultry [14] . The dosage of plant extract was selected based on preliminary pilot trials and previously reported safe phytogenic inclusion levels in poultry feed [5] .

Assessment of Cell-Mediated Immune Response (CMI)

Cell-mediated immunity was evaluated using the 2,4-Dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH) assay, a widely accepted in vivo method for assessing T-cell-mediated immune response in poultry [15] . A featherless area was demarcated on both sides of the abdomen and cleaned with acetone. The right side was sensitized with 2000 µg DNFB dissolved in acetone:olive oil (4:1). The left side served as vehicle control. Fourteen days post-sensitization, birds were challenged with 50 µg DNFB at the same site. Skin thickness was measured at 0, 24, and 48 hours postchallenge using an engineer's micrometer. The mean increase in skin thickness represented DTH response and was interpreted as an indicator of T-lymphocyte activation and macrophage infiltration [16] .

Assessment of Humoral Immune Response

Humoral immunity was assessed by microhaemagglutination assay against sheep red blood cells (SRBC), a classical T-dependent antigen model extensively used in avian immunological studies [3] . Fresh sheep blood was collected aseptically in Alsever's solution and stored at 4 °C for stabilization. The erythrocytes were washed thrice with sterile normal saline by centrifugation at 3000 rpm for 10 minutes. A 7% SRBC suspension was prepared in sterile saline. Six birds from each group were immunized intravenously with 1 ml SRBC suspension. Blood samples were collected on day 10 post-immunization. Serum was separated and heat-inactivated at 56 °C for 30 minutes to inactivate complement proteins. Serial two-fold dilutions were prepared in microtiter plates and incubated with SRBC suspension. The highest serum dilution exhibiting complete haemagglutination was recorded as antibody titre and expressed in log₂ values, reflecting B-cell mediated antibody production [17] .

Hematological Analysis

Heparinized blood samples were collected from the wing vein under aseptic conditions. Total leukocyte count (TLC) was determined using Natt and Herrick's diluting fluid and Neubauer hemocytometer following standard hematological protocols [18] . Differential leukocyte count (DLC) was performed on Leishman-stained peripheral blood smears under oil immersion microscopy. Percentage distribution of lymphocytes, heterophils, monocytes, eosinophils, and basophils was recorded. The heterophil: lymphocyte (H: L) ratio was calculated as an indicator of physiological stress and immune competence [19] .

Immune Organ Weight Index

At the end of the experimental period, birds were humanely sacrificed. Primary and secondary lymphoid organs including spleen, thymus, and bursa of Fabricius were carefully excised and weighed using a precision digital balance. Organ weight index was calculated as:

Relative organ weight is considered a reliable indicator of immune organ development and functional status in poultry [20] .

Statistical analysis

All data were expressed as Mean ± Standard Error (SE) for six birds per treatment group. Statistical analysis was performed using one-way Analysis of Variance (ANOVA) under completely randomized design. When significant differences were observed, Duncan's Multiple Range Test was applied for post-hoc comparison at p < 0.05 significance level [21] . Statistical computations were performed using SPSS software version 26.0.

Results

Cell-mediated immune response (CMI)

Cell-mediated immunity was assessed through measurement of cutaneous hypersensitivity reactions following

Discussion

The present investigation demonstrates that methanolic extract of Tephrosia purpurea exerts a comprehensive immunostimulatory effect in broiler birds, encompassing both cell-mediated and humoral arms of the immune system. The amplified delayed-type hypersensitivity response observed in TPE-treated birds signifies enhanced Tlymphocyte activation and macrophage recruitment hallmarks of a potentiated Th1-mediated immune response. DTH reactions are widely recognized as reliable in vivo correlates of cellular immunity, reflecting antigen-specific T-cell sensitization and cytokine-mediated inflammatory cascades.

The persistence of elevated dermal thickness at both 24 and 48 hours post DNFB challenge suggests that TPE not only accelerates immune activation but may also sustain effector mechanisms. Such sustained responses are indicative of improved immunological vigilance, particularly relevant in poultry production systems where rapid pathogen exposure is common. Contemporary immunopharmacological research has increasingly emphasized plant-derived immunomodulators as alternatives to antibiotic growth promoters in poultry, especially following global regulatory restrictions on antimicrobial use [22,23] . Botanical extracts rich in flavonoids, alkaloids, and phenolic compounds have been shown to enhance macrophage function, nitric oxide production, and cytokine secretion, thereby fortifying host resistance. The elevated haemagglutination titers observed in TPEtreated birds further reinforce the dual modulatory capacity of the extract. Enhanced antibody production implies effective antigen presentation, T-helper cell cooperation, and B-cell maturation. In poultry immunology, HA titers against SRBC serve as classical indicators of humoral competence and vaccine responsiveness. The pronounced increase in titers in the test group suggests that TPE may function as a natural immunological adjuvant, potentially improving vaccine efficacy and long-term protective immunity. Recent studies have highlighted the importance of phytogenic feed additives in optimizing antibody responses and modulating cytokine profiles in broilers (24,25) . Haematological findings provide additional mechanistic insight. The significant elevation in total leukocyte count implies activation of hematopoietic pathways and systemic immune readiness. Increased heterophil proportions, although not statistically significant, align with enhanced innate defense capacity. In avian species, heterophils are critical first-line effector cells, mediating phagocytosis and oxidative burst activity. The observed eosinopenia may reflect modulation of hypersensitivity-related pathways or redistribution of eosinophils during immune activation. Similar immuno-hematological trends have been documented following administration of phytogenic immuno-stimulants in poultry [26] . Interestingly, the absence of significant changes in spleen weight alongside modest increases in thymus and bursa indices suggests that TPE's immunomodulatory action may operate more at the functional and cellular signalling level rather than inducing gross anatomical hypertrophy. 1.00 ± 0.63 0.50 ± 0.22 0.33 ± 0.21 Basophil (%) 0.33 ± 0.21 0.17 ± 0.17 0.17 ± 0.17 Values are mean ± SE. Values in a row with different superscripts differ significantly (p < 0.05).

Conclusion

The present study provides compelling evidence that methanolic extract of Tephrosia purpurea functions as a potent immuno-stimulatory agent in broiler birds. By significantly enhancing delayed-type hypersensitivity responses, elevating hemagglutination antibody titers, and increasing total leukocyte counts, the extract demonstrates a comprehensive capacity to strengthen both cellular and humoral immunity. While changes in lymphoid organ indices were modest, the functional immunological enhancements observed underscore the therapeutic potential of this botanical extract in poultry health management. These findings support the strategic integration of Tephrosia purpurea as a phytogenic immuno-modulator capable of improving disease resistance and reducing dependency on conventional antimicrobial agents. Future investigations employing molecular immunological tools, cytokine profiling, and bioactive compound isolation are essential to fully elucidate its mechanistic pathways and optimize its application in commercial poultry production systems.

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