Electron immunocytochemistry of lymphocyte surface immunoglobulins in the bursa of Fabricius in the pre- and early postnatal ontogeny of chickens

The appearance and localization of surface immunoglobulins on B-lymphocytes of the bursa of Fabricius of developing chick embryos and chicks were studied by immuno-electron-cytochemical peroxidase-antiperoxidase (PAP) method. Ultrastructural characteristics of marked and unmarked lymphocytes of the bursa of Fabricius was shown. The increase in the density of reaction product on PAP-complex in the course of lymphocytes differentiation was established.     

Studies of testosterone-induced involution of the bursa of Fabricius

The present investigation deals with the effect of testosterone on each of the tissue components of the bursa of Fabricius: the endodermal epithelium, the mesenchyme, and the hemopoietic stem cells. Tissue combination experiments between testosterone-treated endoderm and normal mesenchyme and vice versa have shown that the androgen damages irreversibly the bursal epithelium. The latter is not seeded by hemopoietic stem cells and cannot undergo follicle formation when treated with high doses of testosterone. This occurs even if it is associated with a nontreated bursal mesenchyme. On the contrary, associations of testosterone-treated mesenchyme with normal endoderm result in normal bursa histogenesis. By using an original test of viability for lymphoid cells based on the application of the quail-chick marker system, we demonstrate that disappearance of hemopoietic cells in the endoderm results from their expulsion from the bursa and not from their death in situ. The conspicuous effect of testosterone on the bursa of Fabricius can be related to the levels of androgen receptors found in the organ. Typical cytosol androgen receptors are demonstrated in both bursal endoderm and mesoderm, although the amount in the former is higher. The concentration of binding sites in the bursa is >10 times higher than that in other organs such as lung and small intestine whose development is not affected by testosterone, contrasting with glucocorticosteroid receptor (measured by labeling with dexamethasone) found in the same concentration in all tissues.     

Regulating properties of peptides of the thymus and bursa of Fabricius in immunodepression in birds

The intensity of nucleic acids and protein synthesis in the cells of the thymus and the bursa of Fabricius was studied in chickens against the background of an immunodepression induced by administration of hydrocortisone and cyclophosphamide. It was found out that hydrocortisone causes in chicken a marked lowering of the intensity of inclusion of 3H-thymidine, 3H-uridine and 14C-glycine in thymic cells, and cyclophosphamide--in the cells of the bursa of Fabricius. Under the conditions of selective immunodepression the preparations on the basis of the peptides of the thymus (thymalin) and of the bursa of Fabricius (bursilin) regulate the processes of nucleic acids and protein synthesis chiefly in the cells of organs which produce them.     

Sequential Pathology of Experimental Aflatoxicosis in Quail and the Effect of Selenium Supplementation in Modifying the Disease Process

Feeding of aflatoxin B1 @ 1 ppm to 2-week old Japanese quail for a period of 8 weeks produced gross and microscopic changes in the liver, skeletal muscles, heart and bursa of Fabricius. These included fatty changes, bile duct hyperplasia and lymphoid aggregation in liver; haemorrhages in thigh, breast muscles and myocardium; mild depletion of lymphocytes, cystic degeneration and fibrous tissue proliferation in bursa of Fabricius. More or less similar lesions were seen in quail chicks fed on aflatoxin with sodium selenite @ 5 ppm but these were of lesser intensity and appeared at later stages of the experiment thereby indicating that supplementation of selenium had some protective action against the toxic effect of aflatoxin B1 in Japanese quail.     

The effects of trichothecene toxins on the Bursa of Fabricius in day-old chicks.

The effects of T-2 toxin, Fusarenon X (FX), and Nivalenol (NV) on the bursa of Fabricius in the day-old chick were examined. After injections of 5 mg/kg of the mycotoxins into the residual yolk sac, cellular injury was limited at first to the smaller epithelial cells with coarse microvilli, which were located in the central portion of the follicle-associated epithelium. Subsequently necrosis spread out to the periphery. Degeneration and necrosis followed in the lymphoid cells in the lymphoid follicles. The other epithelial components in the follicle were relatively resistant to the mycotoxins. Both FX and NV were less potent than T-2 toxin, although the effects on the bursa of Fabricius were essentially the same. These findings suggest that the follicle-associated epithelium is clearly distinguished from other epithelial components in the bursa of Fabricius in day-old chicks.     

Development of blood testosterone in the embryo and young chick after precocious bursectomy

The effect of bursa of Fabricius on the endocrine function of the chick testes was studied in vivo by comparing plasma testosterone levels from 48 h before hatch to 16 weeks of age in both intact and bursectomized chicken. Early bursectomy was performed at 80 h of incubation. Post surgery survival was low (12% at 1 week). In controls, plasma testosterone levels were found to be low (100-200 pg. ml-1) from 48 h before to 48 h after hatch, then to raise up to a plateau (2,200 pg. ml-1) at 6 weeks. After bursectomy, values were first higher than in intact (210-440 pg. ml-1 from 48 h before/after hatch and 515 vs 300 pg.ml-1 at 3 days) but no difference could be further detected from 1 to 16 weeks of age. It is suggested that, in addition to the effect of androgen on bursa of Fabricius, the later reciprocally influences the gonadotropic axis during the early stage of development.     

Nuclei of lymphocytes of the T- and B-cell lines during normal embryogenesis and after treatment with hydrocortisone (morphometric and probabilistic-statistical parameters)

Lymphoid cells of the thymus and of the Fabricius bursa have been studied in 18-day-old chick embryos, normal and after injection of hydrocortisone on the 11th day of embryogenesis. By means of optical-structural computer analysis, the complex of morphometric and probability-statistic parameters of the nuclei in the lymphocytes are estimated: area of the nuclei, optical density of chromatin, asymmetry coefficient and variance. Normal T-lymphocytes possess less density of the nuclei, greater optical density of chromatin, greater values of negative asymmetry. The complex of these parameters can be used for identification of visually similar lymphoid cells of T- and B-lines. Under hydrocortisone effect structural changes of the nuclei in the thymus and Fabricius bursa lymphocytes of the chick embryo are uniform: increase in the area of the nuclei, decrease in optical density of chromatin, the asymmetry coefficient becomes positive.     

The Cell Cycle Arrest and Apoptosis of Bursa of Fabricius Induced by Low Selenium in Chickens

The purpose of this 42-day study was to investigate the effects of low selenium (Se) on immune function by determining cell cycle and apoptosis of bursa of Fabricius. One hundred twenty 1-day-old avian broilers were randomly assigned to two groups of 60 each and were fed on a low Se diet (0.0342 mg/kg Se) or a control diet (0.2 mg/kg Se), respectively. The relative weight of bursa was significantly decreased in low Se group from 28 days of age in time-dependent manner when compared with that of control group. Cell cycle analysis by flow cytometry showed that low Se caused an increase in G₀G₁ phase cells that corresponded to a decrease in S phase cells in bursa of Fabricius. Ultrastructurally, mitochondria injury and increased apoptotic cells with condensed nuclei were observed. Low Se increased the percentage of Annexin V-positive cells, as measured by flow cytometry, in comparison with that of control group. These data suggested that low Se diet restrained the development of bursa of Fabricius by cell cycle arrest and apoptosis.     

Localization of vasoactive intestinal peptide binding sites in the thymus and bursa of Fabricius of the chick

The purpose of this study was to determine the distribution of VIP binding sites in the thymus and bursa of Fabricius using receptor binding and autoradiographic techniques. Biochemical characterization of 125I-VIP binding sites determined two classes of specific binding sites in both tissues. The dissociation constants determined in the thymus were 1.12 nM and 88.5 nM, and in the bursa were 0.459 nM and 70.8 nM. Autoradiographic localization of 125I-VIP binding sites within the thymus demonstrated specific binding associated with the medullary region of the thymic lobule and the blood vessels in the interlobular and trabecular areas. Within the bursa of Fabricius, high densities of silver grains corresponded with vascular elements in the interfollicular regions, the epithelial border of the plicae, the muscular layer surrounding the organ, and the diffusely infiltrated area near the burso-cloacal duct.     

Effects of feeding a Fusarium toxin-contaminated diet to infectious bursal disease virus-infected broilers on the protein turnover of the bursa of Fabricius and spleen

Two experiments were carried out to examine the effects of feeding an uncontaminated control diet (CON) or a Fusarium toxin-contaminated diet (FUS; 10.7 mg deoxynivalenol [DON]/kg diet) to growing broilers, which were either uninfected or infected with infectious bursal disease virus (IBDV) beginning at 1 day post hatch. Broilers had been infected at three weeks post hatch with either a classical virulent infectious bursal disease virus (IBDV-IM, Exp. 1) or a very virulent IBDV (vvIBDV, Exp. 2) strain. The effects of the DON-contaminated diet in combination with the virus-infection on the bursa of Fabricius and spleen were determined at 3 and 6-7 days post infection. The transient development of the bursa oedema and the bursa atrophy was not significantly affected by the diet after infection with the different IBDV-strains. The histopathological lesions were more severe in IBDV-IM-infected birds at 6 days post infection when additionally exposed to the FUS diet as compared to the FUS-free feed. Most parameters of the bursa of Fabricius and spleen protein turnover (e.g. fractional protein synthesis rate, protein, DNA and RNA content and derived indices) were significantly and interactively influenced by infection and stage of infection. The vvIBDV-infected birds responded with a more pronounced depressing effect on the fractional protein synthesis rate after feeding the DON-containing FUS diet when compared to their IBDV-IM-infected counterparts, where the opposite effect was observed. It can be concluded that feeding a FUS diet to IBDV-infected broilers might modulate the virulence-dependent pathogenesis of an IBDV infection.     

Immunomodulatory roles and functional analysis of pre-B lymphocyte DT40 cells with the bursal-derived BSP-II treatment

The bursa of Fabricius, the acknowledged central humoral immune organ, is vital to B cell differentiation. However, the regulatory function of the bursal-derived peptide on avian B cell proliferation has not been reported. BSP-II is a recently reported bursal-derived bioactive peptide. In this paper, 75 days-old chicks were twice subcutaneously immunized with BSP-II and inactivated avian influenza virus (AIV, H(9)N(2) strain). It was proved that BSP-II induced a strongly AIV-specific HI antibody production in the immunized chicks. Also, BSP-II could enhance avian pre-B lymphocyte DT40 cell viability. To investigate the global patterns of gene expression in DT40 cells after BSP-II treatment, gene microarray was carried out. It was identified that the differentially expressed genes were involved in various pathways, of which six pathways were associated with signaling transductions, including ErbB signaling, MAPK signaling, Toll-like receptor signaling, Notch signaling, mTOR signaling, and Wnt signaling. Finally, RT-qPCR was used to confirm the microarray expression data. These results indicated the molecular basis of pre-B lymphocyte viability with BSP-II treatment, which provided a potential mechanism of the bursa of Fabricius on pre-B lymphocyte viability, differentiation, and development. These results are valid for the mechanism of the bursa of Fabricius on B lymphocytes development.     

Biochemical characterization of the progesterone receptor in the chick bursa of Fabricius

In a previous work we demonstrated estrogen-inducible progesterone binding sites in the bursa of Fabricius. In the present study these were characterized and compared to the progesterone receptor (PR) in the chick oviduct. When the size of the binding sites was analyzed with sucrose gradient centrifugation, 2 peaks of bound progesterone were obtained. The sedimentation coefficients of the peaks were 8-9 S and 3-4 S. In size exclusion HPLC only 1 peak was seen with a size corresponding to the 8-9 S in the sucrose gradient. The Stokes radius was 7.7 nm. When the ionic strength was elevated or CaCl2 was added, smaller steroid binding forms were detected. The sizes of these progesterone binding molecules at low and high ionic strength and in the presence of CaCl2 were equal in bursa and oviduct when analyzed with HPLC. The Stokes radii of these forms were 5.6 nm in high salt and 2.1 nm with CaCl2. The steroid binding components in the bursa cytosol eluated as 2 peaks from the DEAE column with KCl gradient. The peaks corresponded to the so-called A and B components in the chick oviduct. In the presence of molybdate, bound progesterone eluated as one peak from DEAE in both oviduct and bursa. The progesterone binding capacity was shown to be heat labile with equal half-lives in the bursa and the oviduct. Progesterone and ORG 2058 had a high affinity for the binding site and their binding was specific for progestins. It is concluded that the estrogen-inducible progesterone binding site in the bursa of Fabricius resembles the oviductal progesterone receptor in structural and binding properties.     

PANCREAS OF ASELLI IN SOME SPECIES OF THE SHREWS (SOREX ARANEUS & NEOMYS FODIENS) AS AN ANALOGUE OF THE BURSA OF FABRICIUS IN BIRDS

No organ equivalent to the bursa of Fabricius in birds, which is responsible for the production of B cells, has been found in mammals thus far. It has been suggested that Peyer's patches and the appendix along with the bone marrow play this role. In this study, the gland of Aselli of shrews was examined, whose function has not yet been clarified. The results of the study of the gland of Aselli in common and water shrews suggest this gland to be the key lymphoid organ analogous to the bursa of Fabricius. However, unlike the bursa of Fabricius, the gland of Aselli seems to fulfil two functions. It not only produces B cells, but also is the site of their terminal differentiation into plasma cells.     

Effects of Dietary Selenium on Selenoprotein W Gene Expression in the Chicken Immune Organs

Selenoprotein W (SelW) is expressed in the immune systems of mammals. However, its pattern of expression in the immune organs of birds is still unclear. To investigate the distribution of SelW and effects of dietary Se levels on the SelW mRNA expression in the immune organs of birds, 1-day-old male chickens were fed either a commercial diet or an Se-supplemented diet containing 0.601, 1.058, 1.514, or 2.427?mg Se per kilogram, and 1.0, 2.0, 3.0 or 5.0?mg sodium selenite per kilogram for 90?days. The immune organs (spleen, thymus, and bursa of Fabricius) were collected and examined for Se content and SelW mRNA levels. The mRNA expression of SelW was detected in all the tissues. Although Se content was the highest in the spleen, the remarkable stability of the SelW mRNA level was observed in this organ during different times of dietary Se supplementation. Se-supplemented diet can make the SelW expression levels higher within a certain range in thymus and bursa of Fabricius. The present study demonstrates that SelW is widely expressed in immune organs of birds and that Se-supplementation of the feed increases SelW expression in the thymus and the bursa of Fabricius.     

Effects of estradiol on the development of the bursa of Fabricius in Japanese quail

Effects of androgens on the development of the bursa of Fabricius are better understood than those of estradiol, despite the known sensitivity of the bursa to estradiol early in embryogenesis. The goal of this study was to determine the effects of one-time yolk injections of estradiol at day 4 of incubation on the development of the bursa and spleen as indices of treatment effects on the immune system. Follicle size and numbers in hatchling bursas were significantly reduced at 50 and 500 microg/egg, respectively. Additionally, distorted plicae and thicker epithelial layers surrounding the plicae were observed in day-old chicks at the same treatment levels. Adult bursas from birds embryonically exposed to estrogen were significantly larger than controls, suggesting an inhibition of natural bursal regression. Although estradiol altered the development of the bursa, the spleen appeared to be unaffected. The observed effects of estradiol on the development of the bursa indicate that this lymphoid organ may be a target for developmental disruption by estrogenic endocrine disrupting chemicals, though long-term consequences of embryonic exposure on immune function remain unknown.     

Hyperpigmentation Results in Aberrant Immune Development in Silky Fowl (Gallus gallus domesticus Brisson)

The Silky Fowl (SF) is known for its special phenotypes and atypical distribution of melanocytes among internal organs. Although the genes associated with melanocyte migration have been investigated substantially, there is little information on the postnatal distribution of melanocytes in inner organs and the effect of hyperpigmentation on the development of SF. Here, we analyzed melanocyte distribution in 26 tissues or organs on postnatal day 1 and weeks 2, 3, 4, 6, 10, and 23. Except for the liver, pancreas, pituitary gland, and adrenal gland, melanocytes were distributed throughout the body, primarily around blood vessels. Interaction between melanocytes and the tissue cells was observed, and melanin was transported by filopodia delivery through engulfed and internalized membrane-encapsulated melanosomes. SFs less than 10 weeks old have lower indices of spleen, thymus, and bursa of Fabricius than White Leghorns (WLs). The expression levels of interferon-γ and interlukin-4 genes in the spleen, and serum antibody levels against H5N1 and infectious bursal disease virus were lower in SF than in WL. We also found immune organ developmental difference between Black-boned and non-Black- boned chickens from SFs and WLs hybrid F2 population. However, degeneration of the thymus and bursa of Fabricius occurred later in SF than in WL after sexual maturity. Analysis of apoptotic cells and apoptosis-associated Bax and Bcl-2 proteins indicated that apoptosis is involved in degeneration of the thymus and bursa of Fabricius. Therefore, these results suggest that hyperpigmentation in SF may have a close relationship with immune development in SF, which can provide an important animal model to investigate the roles of melanocyte.     

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. II. Immune response of bursectomized chicks and chimeras and post-natal rejection of the grafted quail bursas

Two methods to bursectomize chick embryos before hemopoietic cell seeding of the bursa of Fabricius were compared in this work: section of the tail region at E3 including the presumptive bursal territory, and selective removal of the bursa at E5. Hatching ability is better with the former method, but survival rate and effectiveness of bursectomy are favored with the second, novel technique. Moreover, selective removal of the bursa at E5 can be followed by in situ engraftment of a quail bursa and construction of quail-chick bursal chimeras. The immune response of bursaless birds and bursal chimeras has been studied. Total absence of the bursa does not prevent a few B cells from differentiating and nonspecific Ig (IgM and/or IgG) from being secreted. As reported previously, bursaless birds, however, are unable to mount an immune response by producing specific antibodies. This immune function is restored by the graft of a quail bursa. The microenvironment of the bursa, although heterospecific, allows the expansion of the B cell population and generates the repertoire of the B cell antigen receptors. This process takes place during late embryonic and early postnatal life because the grafted quail bursal stroma is subjected to immune rejection from 2 to 3 wk after birth in all chimeras, which are, however, perfectly immunocompetent.     

Cholinesterase activity in quail primary lymphoid organs

The present histochemical study was carried out to analyze the distribution and topography of acetylcholinepositive nerve fibers in the thymus and bursa of Fabricius of quails. The AChE-positive nerve fibers were demonstrated by direct thiocholine histochemical method. Nerve fibers present in the thymuses form periarterial nerve plexuses located mostly in the interlobular septa and on the cortico-medullary junction. Vessels-independent nerve fibers occur also in the parenchyma of thymic medulla, but rarely in parenchyma of the cortex. Within the connective tissue between the bursa of Fabricius and the wall of proctodeum we observed conspicuous AChE-positive ganglia, often in close relationship to greater arteries. Within the wall of bursa of Fabricius, AChE-positive nerve fibers create nerve plexuses around arteries. We observed a close relationship between lymphoid follicules in bursal submucosa and mucosa and AChE-positive nerve fibers. Nerve fibers create a ring around lymphoid follicles, but do not penetrate into the germinal center of the follicle. Arteries inside quail thymuses and bursae of Fabricius contain rich AChE-positive nerve plexuses, when compared to the veins, which have a very poor presence of AChE-positive nerves. According to lesser presence and decreased density of AChE-positive nerve fibers in older animals, we described age-dependent changes in both quail primary lymphoid organs.     

Ontogeny of oestrogen-sensitive mesenchymal cells in the bursa of Fabricius of the chick embryo. An immunohistochemical study on progesterone receptor

The expression of progesterone receptor (PR) and its induction by oestradiol during the embryogenesis of the chick bursa of Fabricius (BF) were studied by immunohistochemistry using three different polyclonal antibodies to the chicken oviduct PR. Mesenchymal cells of the cloacal area surrounding the bursa primordium in controls (without exogenous oestrogen) express the PR between 9 and 11 days of incubation. In the same cells, PR was induced experimentally by oestradiol at 9 days. Mesenchymal cells in the bursa did not express PR after oestradiol treatment before the age of 11 days. The PR was not inducible in the bursal epithelium or in haemopoietic cells. None of the bursal cells expressed the PR to a detectable level during embryonic life without exogenous treatment. Some haemopoietic cells showed strong artefactual staining in their nuclei. It is concluded that (1) the embryonic bursa of Fabricius is a sex-steroid-sensitive organ, (2) exogenous oestradiol is able to induce progesterone receptor in the mesenchymal cells, but (3) the PR is not expressed without exogenous oestrogen. This indicates that the PR becomes oestrogen inducible well before it is naturally expressed during sexual maturation and that the level of endogenous oestrogen during embryonic life is not high enough to affect the bursa significantly.