The Rainbow Rooster is a low-maintenance, dual-purpose breed that matures between 4 to 6 monthsand lays approximately 160-180 eggs over 16 months (Singh et al., 2021). Poultry farming, which includes both layer and broiler production, contributes to food security while also serving as a profitable source of income (Toorand Goel, 2024). Backyard poultry farming, especially in rural and hilly areas of India, holds promise for addressing nutritional deficiencies, poverty reduction, women's empowerment and self-employment (Islam et al., 2020). In Mizoram, a significant gap exists between the demandand supply of meat and eggs, with a shortage of 1540.559 tonnes of meat and 1992 lakh eggs (Economic Survey of Mizoram, 2021-2022). Bridging this gap requires farmer training in scientific poultry practices, including vaccination, feed managementand housing (Singh et al., 2021). Immunity is vital for poultry health, with the thymusand Bursa of Fabricius being primary lymphoid organs (Glick et al., 1956). Bursa of Fabricius is closely linked to the cloaca and digestive system. It is generally recognized as the primary lymphoid organ in chickens and is a crucial organ for the immune system to respond to environmental antigens (Ifrah et al., 2016). It appears by Day 5 of embryonic development, is essential for B-cell maturation and begins involution around 20 weeks (Fellah et al., 2014). Bursa removal impairs B-cell function and antibody production (Glick et al., 1956). Infectious Bursal Disease (IBD) is caused by the Infectious Bursal Disease Virus (IBDV), a double-stranded RNA virus belonging to the family Birnaviridae. The virus specifically targets bursal B cells, leading to extensive lymphocyte destruction and resulting in lymphopenia (Shinde et al., 2021). It primarily affects 3 to 6-week-old chicks, damaging B cellsand causing immunosuppression (Caston et al., 2008; Dey et al., 2019; Pandey et al., 2021). The disease can lead to 100% morbidityand up to 60% mortality, depending on the strain (Jackwood, 2019). Vaccination is critical in controlling diseaseand boosting poultry productivity (Marangonand Busani, 2007).

Collection and preparation of samples
 
Chickens from each group were sacrificed by the cervical dislocation method (Jaksch, 1981). The foodand water were withheld for two hours prior to their slaughter. After the chickens were slaughtered, the feathers were physically removedand the bursa of Fabricius was retrieved via ventral abdominal dissection. The samples were cleaned with normal saline and blotted using blotting paper. The organs were observed critically. After collecting gross data, the bursa was preserved in 10% neutral buffered formalin and prepared for paraffin wax embedding. The samples were dehydrated in increasing alcohol concentrations for two hours. The tissue was then embedded in paraffin wax after clearing. Sections were cut to a thickness of 5-6 mm using a semi-motorized rotary microtome. Subsequently, the samples were treated with various stains to facilitate histological analysis (Bancroftand Cook, 1994).
 
Location of experiments
 
The present research work was conducted from April 2022- April 2023 in the Department of Anatomyand Histology, College of Veterinary Sciencesand Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram, India.
 
Experimental design
 
This study involved 57 chickens representing different gendersand stages of development. The chickens were divided into two groups: 30 in the control group (C)and 27 in the treatment group (T). The treatment group received an oral dose of a commercially available live IBDV vaccine on days 14and 21. Each bird was given one drop containing at least 103 PFU of the intermediate strain of IBD virus per dose. Six chicks from each group (C1 and T1) were maintained for blood sample collection, while three birds from each group (C2and T2) were sacrificed at 4-week intervals from 0-28^th weeks for collection of the Bursa. The experiment was ethically approved by the Institutional Animal Ethics Committee (IAEC), vide Approval Reference Number CVSC/CAU/IAEC/21-22/P-4 dated 16.11.2022.

Histomorphology
 
The Bursa of Fabricius comprises of three distinct layers: the tunica serosa, tunica muscularisand tunica mucosa (Fig 1). The tunica serosa includes mesothelium, subserosal connective tissue, blood vesselsand nerve endings. The tunica muscularis contains major blood vessels, nerves and two layers of smooth muscle fibers. The innermost tunica mucosa features the lamina epithelialis and lamina propria, the latter forming bursal folds that enclose lymphoid follicles. These findings align with Ebru et al., (2015), Tamilselvan et al., (2017) and Dahariya et al., (2020), while Jain et al., (2010), Fangand Peng (2014) and Penchev (2020) reported four tunics, indicating possible breed differences. These follicles are organized into a cortexand medulla, separated by a clearly defined corticomedullary border visible by the 8th week in the control groupand by the 4^th week in the treatment group similar to findings by Kempashi et al., (2017), Ayman et al., (2020) and Wu et al., (2021). The cortex contains lymphocytes, lymphoblasts, macrophagesand plasma cells, while the medulla comprises reticular cells, macrophagesand plasma cells (Fig 2). The cortex stains more intensely due to the higher density of small lymphocytes consistent with Sultana et al., (2011), Yadav et al., (2020) and He et al., (2015). Medullary plasma cells exhibit prominent rough endoplasmic reticulum and large Golgi regions as also noted by Yadav et al., (2020). Secondary follicle or germinal center formation began at 4th weeks in the treatment group and at 8th weeks in the control group. The bursal folds are lined with two epithelial types: Follicle-associated epithelium (FAE), made of simple columnar cellsand Inter-follicular epithelium (IFE), composed of pseudostratified columnar cells (Fig 2), supporting findings by Romano et al., (1996), Kanasiya et al., (2017) and Kadam et al., (2020), Wardand Middleton (1971) and Ebru et al., (2015) observed variations in cell types across species. Intraepithelial lymphocytes (IELs) were unevenly distributed, with higher density in the treatment group, consistent with Ayman et al., (2020). The epithelium contained columnar, basaland goblet cells, as described by Onyeanusi et al., (1993) and Kadam et al., (2020), while Kanasiya et al., (2017) and Yadav et al., (2020) noted four cell types in Kadaknath chickens, suggesting breed-specific variation.





In this study, collagen fibers (red) were identified in the connective tissue of the tunica muscularisand the lamina propria around the follicles (Fig 3) using the van Gieson’s staining (van Gieson, 1889). These findings align with Kanasiya et al., (2017) in Kadaknath chickens, Dahariya et al., (2020) in Hansli and Vencoob broiler chickenand Udoumoh et al., (2022) in broilers. At day 0, these fibers were sparse but increased with age in both control and treatment groups. Although the distribution pattern remained similar across groups, collagen fiber presence became more prominent by the 24^th week, showing greater abundance compared to the 0-day group. In contrary, Jain et al., (2010) noted higher fiber density in Vanaraja chicks compared to CARI Shyama growers, indicating possible breed-specific differences. Reticular fibers (black) were observed alongside collagen fibers using the Gomori’s staining method (Gomori, 1937) in the loose connective tissue of the tunica muscularis and around the follicles in the lamina propria (Fig 4). These finding were consistent with findings by Kanasiya et al., (2017) in Kadaknath birds and Deka et al., (2020) in Pati ducks. Although less dense than collagen fibers, reticular fibers increased with age in both control and treatment groups. However, no variation in their distribution was noted within the same age group, indicating no influence from IBDV vaccination. Hart’s staining (Mallory, 1961) revealed elastic fibers (blue-black to black) in the bursa of Fabricius, primarily around blood vessels in the tunica mucosa (Fig 5) consistent with findings by Kanasiya et al., (2017) in Kadaknath chickens, Dahariya et al., (2020) in Hansli and Vencoob broilers. No differences were observed between control and treatment groups of the same age, suggesting the IBDV vaccine had no effect on tissue fibers. PAS-Alcian Blue staining at pH 2.5 (Mowry and McManus, 1963) revealed weak magenta staining, indicating low mucopolysaccharide presence. Both the epitheliumand tunica mucosa showed weak PAS positivity across all age groups in both control and treatment birds (Fig 6). These findings were similar with Sugimura et al., (1975) and Kempashi et al., (2017). Methyl Green Pyronin staining revealed positive detection of DNA (green-blue)and RNA (red) in the follicular cells of all experimental birds (Fig 7). Both control and treatment groups showed consistent DNAand RNA positivity across all age groups. These findings align with Gulmezand Aslan, (1999) in native geese. Bielschowsky’s staining identified black-stained nerve fibers, including axis cylindersand dendrites, in the tunica muscularis of the bursa of Fabricius (Fig 8). These fibers were present in all experimental birds from both control and treatment groups, regardless of age, consistent with Deka et al., (2020) in Pati ducks.












 
Histomorphometry
 
The number of follicles per plica increased with age until the 12^th week, then declined in both groups (Fig 9). The treatment group showed a significantly higher follicle count at 8^th and 12^th weeks, with the highest number observed at 12^th weeks. Jain et al., (2010) observed a higher number of follicles in CARI Shyama chicksand Vanaraja growers. Similarly, Kanasiya et al., (2017) reported the highest follicle count at 8^th weeks in Kadaknath chickens, while Hashimoto and Sugimura (1976) recorded a peak at 13^th weeks in White Pekin ducks. In contrast, Ayman et al., (2020) documented continuous follicular growth in Sonali chickens. Notably, a significant increase in follicle count at the 8thand 12^th weeks in the treatment group may be attributed to an elevated immune cell response. Plicae length (Fig 10) and breadth (Fig 11) increased significantly up to the 12^th week, followed by a gradual decline. Peak lengthand breadth occurred at 12 to 16^th weeks in the control group and 8 to 12^th weeks in the treatment group. Ayman et al., (2020) reported continuous growth in Sonali chickens up to 56 days. A highly significant difference of plical length between the control and treatment groups was observed from 4^th to 12^th weeks possibly due to increased immune cells. The maximum height of the lining epithelium (Fig 12) in the control group, was observed between the 8^th and 12^th weeks, while in the treatment group, it peaked between the 4^th and 12^th weeks. A significant difference was noted at 4^th weeks of age in the treatment group compared to the control. The thickness of the cortex (Fig 13) increased until the 12^th week, then gradually declined in both groups, showing significant differences. A notable difference between the groups was observed in the treatment group at the 8^th and 12^th weeks likely due to bursal maturation and increased immune cells. The control group showed maximum medullary breadth (Fig 14) at 12^th weeks, while the treatment group peaked between 8^th to 12^th weeks. The breadth increased until the 12^th week, then gradually declined, with a significant difference between the two groups at the age of 16^th and 20^th weeks. Ayman et al., (2020) observed continuous growth of lining epithelium, cortex thickness and medullary growth in Sonali chickens from 0-56 days.

From the study, we can conclude that, the involution of the bursa was prolonged in the treatment group. There was early development of secondary follicles in the bursa of Fabricius in the treatment group. A significant increase in lymphocyte infiltration was observed in the bursal follicles of the treatment group. The treatment group showed a higher antibody response compared to the non-vaccinated control group.

The present study was supported by the College of Veterinary Sciencesand Animal Husbandry, Mizoram, Central Agricultural University. The authors sincerely express their gratitude for providing the necessary facilities and support for this research.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal Care and Handling Techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.