Introduction

Unlike other vertebrates, avian species have two discrete primary lymphoid organs, the thymus in which T-lymphocytes develop and responsible for cell mediated immunity (CMI) and the bursa of Fabricius, the primary site for B-lymphopoiesis which is responsible for humoral immunity (HI) (Glick, 1964 and Warner, 1967). The bursa of Fabricius in chicken is located at the dorsal portion of cloaca and connects with the cloaca by the bursal duct at its junction with the colon (Glick, 1964).It has been recognized that the bursa also functions as a peripheral gut-associated lymphoid organ. Antigens presented via the cloaca and bursal lumen can stimulate specific antibody production by bursal lymphocytes (Lupetti et al., 1984). Thus, the bursa plays a role in local gut immunologic defence. A critical component of the local bursal response is the surface epithelium of the bursa overlying the medullary region of the lymphoid follicles – the follicle-associated epithelium (Houssaint et al., 1986).

The chicken is a foundational model for immunological research and continues to be a valuable animal model for insights into immune function. Hence, the present research work is aimed to understand the changes in histoarchitecture of the bursa of Fabricius in different age groups using light microscopy.

Materials and Methods

Collection of Bursa

Twenty four birds for the present study were procured from Institute of Poultry Production and Management, Madhavaram Milk Colony, Chennai. Bursal tissue were collected from four different age groups viz., day-old, second, fourth, and sixth week. Six birds each were utilized from each age group for histomorphological study. The bursa was removed immediately after high cervical dislocation and fixed for routine paraffin embedding and for light microscopy (Kannan et al., 2015).

Methods

Tissue pieces were collected from bursa of Fabricius and were rinsed in normal saline and fixed in 10 percent neutral buffered formalin. The fixed tissues were dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin wax. Bursa samples were processed for paraffin embedding and sections of 3-5 µm thickness were cut using microtome and utilized for haematoxylin and eosin staining (Bancroft and Gamble, 2008). All chemicals, substrates and kits required for histochemical staining methods were of analytical grade and procured from Himedia®.

Results and Discussion

The wall of the bursa of Fabricius was made up of three tunics. The outermost, tunica serosa was thin and enclosed the whole organ. The middle tunica muscularis, consisted of an outer circular and an inner longitudinal layer of smooth muscle fibres and the longitudinal layer was found to enter the central core of the mucosal plicae. The innermost tunica mucosa consisted of a lining epithelium and lamina propria filled with lymphoid follicles (Fig.1). This is in total agreement with the findings of Hodges (1974), King and McLelland (1984), and Onyeanusi et al. (1993) in domestic fowl. Whereas, Gulmez and Aslan (1999) reported that in native geese, the tunica muscularis was made up of an outer layer of longitudinal and inner circular smooth muscle fibres. The tunica mucosa was thrown into folds, the primary plicae which branched out to give rise to secondary plicae as per Sabiha et al. (1999) in the bursa of Japanese quail. The lining epithelium was made up of pseudostratified columnar epithelium. Three types of cells were found in the epithelium.

Fig.1: Photomicrograph of panoramic view of the bursa of Fabricius of four week-old chicken; TM-Tunica muscularis; C-Cortex; M-Medulla; E-Epithelium; LF-Lymphoid Follicle (H&E x 40)

Type-I were columnar cells with round or oval nucleus. Type-II cells were basally placed with round nucleus. Type-III was goblet cells found among the columnar cells (Fig.2).

Fig.2: Photomicrograph of the bursa of Fabricius of six week-old chicken showing three types of cells in the epithelium; C-Columnar cells. B-Basal cell; G-Goblet cell; Bm-Basement membrane (H&E x 1000)

The epithelium covering the plicae were divided into two types viz., follicle associated epithelium (FAE) or epithelial tuft with pale columnar cells which was in direct contact with medulla of the lymphoid follicle and the interfollicular epithelium (IFE) covering the remaining part of the plicae consisting of darkly stained columnar cells (Fig.3) as reported by Ackerman and Knouff (1959) and Hodges (1974) in chicken.

Fig. 3: Photomicrograph of the bursa of Fabricius of four week-old chicken showing the follicle associated epithelium (arrow heads) and interfollicular epithelium (arrow); M-Medulla; C-Cortex(H&E x 40)

The lymphoid follicle present in the lamina propria was of various shapes and sizes. Each follicle consisted of an outer thin cortex and an inner large medulla, separated by a layer of epithelial cells with distinct basement membrane and a thin layer of capillary network (Fig.4) confirming the reports of Hodges (1974). The cortex contained densely packed lymphoblasts and lymphocytes with few macrophages, while medulla was made up of lymphocytes and loosely arranged reticulo-epithelial cells (Fig.5). Similar findings were reported by Mazzone et al. (2003) in White Leghorn chicken.

Fig. 4: Photomicrograph of the bursa of Fabricius of six week-old chicken showing cortex, medulla, epithelium in the cortico- medullary junction (arrow heads) and interfollicular connective tissue (arrow); M-Medulla;C-Cortex (H&E x 400)

Fig. 5: Photomicrograph of the bursa of Fabricius of six week-old chicken showing reticulo-epithelial cells (arrow head) in the medulla of lymphoid follicles; LL, ML and SL- Large, Medium and Small sized lymphocytes(H&E x 1000)

Summary

In the present study, the histological structure of bursa of Fabricius of day-old and two, four and six week-old chicken was similar except an increase in the size of the lymphoid follicles and height of the epithelium. The demarcation between cortex and medulla was clearly evident with the advancement of age.

Acknowledgement

Authors are highly thankful to the Dean, Madras Veterinary College, Chennai – 600 007 for providing necessary financial support.

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