MY THESIS...

Thesis Title :“ONTOGENY OF LYMPHOID ORGANS CHICKEN EMBRYOS (GALLUS DOMESTICUS)”.

SUMMARY AND CONCLUSION

            During the process of evolution living creatures developed various defence mechanisms to protect themselves from the onslaughts of physical, chemical and biological adversaries. The complex immune system constitutes one of the most important mechanisms. Proper development and intactness of the immune system is thus, considerably significant not only for the well being but also for the survival of the living being. Any abrogation in normal immunological functions renders the animals susceptible to various causative agents resulting into reduced production and/or loss of the animals. Hence, the study of development, compartmentalization and differentiation of the components of the immune system is of immense significance, especially in the fast changing environmental scenario.

          The present research plan was formulated to study the development, structural organization and functional status of immune system in relation to the gross morphological, histoarchitectural and histochemical development of the lymphoid organs (thymus, bursa of Fabricius and spleen). The investigation was carried out on two embryos of each 6 and 11 day, ten embryos of each 14 and 18 day and twenty embryos each of 16 and 21 day. To access the immunity in developmental stages of embryo along with lymphocyte blastogenesis and antibody titres, different cells (T-cells, pan B-cells, IgG secreting cells, IgM secreting cells and mononuclear phagocytic cells) were detected in the lymphoid organs by immunoperoxidase technique.

          Thymus, bursa and spleen weighed 22.0 mg, 9.8 mg and 1.9 mg at 16^th and 98.3 mg, 44.7 mg and 13.2 mg at 21^st day of incubation. Length x width of thymus was 18.5 x 3.5 mm, 24.5 x 4.5 mm and 32.5 x 6.5 mm at 16^th, 18^th and 21^st day of incubation. Similarly diameter of bursa and spleen was recorded to be 3.0 mm and 2.0 mm at 14^th, 3.5 mm and 2.5 mm at 16^th, 4.5 mm and 3.0 mm at 18^th and 6.5 mm and 4.5 mm at 21^st day of incubation, respectively.

          Histologically, in 6 and 11 day old embryos, thymus was a group of epithelial cords adjacent to the jugular vein. At 14^th day some lymphocytes and blood vessels appeared in thymus. By the 16^th day stroma of thymus started differentiating into different lobules with the appearance of thin connective tissue septae. The lobules were well developed at 18^th day. At 21^st day lobules were differentiated into outer darker cortex and inner lighter medulla, with many lymphocytes in the cortical region. At 11^th day bursa was a rounded organ with a lumen in center. At 14^th day plicae were projecting into lumen, with the evidence of epithelial buds formation. By the 16^th day number of lymphocytes increased in epithelial buds along with the appearance of reticular fiber network. By the 18^th day the shape of the plicae changed from leaf like to club shaped with the formation of secondary plicae. At 21^st day there was a heavy increase in number of bursal follicles encircled by collagen and reticular fibers. But follicles were not differentiated in cortex and medulla. Plicae towards the luminal side were lined by low columnar or pseudostratified columnar epithelium. Spleen at 6 and 11 day appeared as a mass of mesenchymal cells and some sinusoids containing RBC’s and at 14^th day had venous sinuses lined by littoral cells and few arterial blood vessels. At 16^th day, the reticular fibers surrounding the arterial blood vessels extended to the adjacent areas. Hematopoietic cells appeared around the blood vessels. After 16^th day, the reticular fiber network became more extensive. But lymphoid nature of white pulp was not established till the 21^st day of incubation.

          In histochemical studies, activity of acid phosphatase was found in thymus at 18^th and 21^st day of incubation. Activity of cholinesterase was seen at 18^th day. A weak activity of ATPase was also found at 21^st day. Activity of glucose-6-phosphatase, lipase, 5`-nucleotidase and alkaline phosphatase were absent in the thymus. Lipids, carbohydrates and mucopolysaccharides were also not observed in the thymus. In bursa, a mild activity of acid phosphatase at 18<sup>th</sup> day and an intense activity at 21<sup>st</sup> day was observed. Activity of cholinesterase was seen only at 18<sup>th</sup> day of incubation. ATPase, glucose-6-phosphates, 5`-nucleotidase and alkaline phosphatase activities were absent in the bursa. A little PAS positive material was found in the lining of plical epithelium and in center of some of the bursal follicles at 18^th day, which at 21 the day increased in amount in epithelial lining. Lipids and mucopolysaccharides were undetectable in the bursa. In spleen, at 18^th day, acid phosphatase activity was found mainly in endothelium of blood vessels but at 21^st day intense generalized activity was seen in the organ. Activity of cholinesterase was found in endothelium of blood vessels of spleen at 16^th, 18^th and 21^st day of incubation. Activity of ATPase, glucose-6-phosphatase, lipase, 5`-nucleotidase, and alkaline phosphatase was not found in the spleen. Similarly lipids, carbohydrates and mucopolysaccharides were also not found in the spleen.

          In immunological studies, the mean delta OD in lymphocyte stimulation was 0.055 and 0.1085 at 16th day and 1.662 and 1.585 at 21 day for LPS and Con-A stimulated cultures, respectively. The mean ELISA values for IgG and IgM immunoglobulins were 2.356 and 2.241 at 16^th day and 2.172 and 2.013 at 21^st day of incubation, respectively. Immunohistochemical studies revealed that thymus was having mainly T-cell, but also a few B-cells. In addition, mononuclear phagocytic cells and IgM secreting cells were seen at 16^th as well as at 21^st day. IgG secreting cells were found only at 21^st day of incubation. In bursa the main cell type was B-cells. Mononuclear phagocytic cells, IgM secreting cell were also found at 16^th and 21^st day. IgG secreting cells were found in bursa only at 21^st day of incubation. Few T-cells were also found in the bursa. The T-cells, B-cell, IgM secreting and IgG secreting cells formed different ‘colonies’ in spleen while, mononuclear phagocytic cells were scattered all over the organ. IgG secreting cells in spleen appeared at the 21^st day of incubation only.

          From the above findings, it can be concluded that the thymus was largest organ, of all the three lymphoid organs studied, at all the stages of development. Percent increment in weight was maximum in thymus followed by spleen and bursa during 18-21 day of incubation. In proportion to the body weight of embryo thymus was ranked first followed by bursa and spleen. However, percent increment in size of spleen was highest followed by bursa and thymus. Histologically, the development of thymus was delayed when compared with older literature. However, at 21^st day of incubation thymus was fully formed, with well differentiated cortex and medulla of thymic follicles. The development of bursa was also delayed. At 21^st day of incubation bursal follicles were not divided into cortex and medulla. But, B-cells were observed in bursa by immunoperoxidase and these cells were in a functional state as indicated by lymphocyte stimulation test. The development of spleen was not much delayed when compared with older literature. The establishment of lymphoid nature of white pulp did not occur during the prehatch period. Lymphocyte stimulation test depicted that the B-cells and T-cells took up their function only at 21^st day but not at 16^th day of incubation. The ELISA indicated that IgG and IgM antibodies of maternal origin were present in blood of the embryo at both 16 and 21 day of incubation. 

          Further investigation on this aspect can be carried out by accessing the status of cytokines during embryonic and neonatal life of chicken along with the extensive study of various cell surface markers. Functional status of immune system and presence of maternal immunity during late embryonic and neonatal stages of chicken can lead to a more successful way of immunization in early stages of bird’s life.