Survival and function of bursa-derived cells in bursectomized chickens

Chicken given heterologous antibodies to μ chains as embryos and again following bursectomy at hatching were rendered permanently agammaglobulinemic, whereas treatment with anti-μ alone resulted in transient hypogammaglobulinemia. These and other observations indicate that sites other than the bursa of Fabricius are unable to serve as sources of immunoglobulin-producing cells. Agammaglobulinemia did not develop in birds bursectomized at 1, 14, or 35 days after hatch during an observation period of 21–125 wk. Some bursectomized birds developed 10 × normal IgM levels but were very deficient in IgG; this pattern persisted throughout life. B lymphocytes, identified by surface immunoglobulins in high density, were permanently deficient in bursectomized animals despite development of hypergammaglobulinemia and high levels of antibodies to ferritin following hyperimmunization. In experiments employing embryonic bursectomy, the level of the circulating pool of B lymphocytes was shown to be dependent upon the time allowed for the bursa to function before removal.     

Immune capacity of the chicken bursectomized at 60 hr of incubation: surface immunoglobulin and B-L (La-like) antigen-bearing cells

Chickens were surgically bursectomized at 60 hr of incubation before the bursal anlage appears. Completeness of the bursectomy was confirmed at autopsy at 10 wk of age. These embryonically bursectomized (Bx) chickens are known to produce immunoglobulins of IgM, IgG, and IgA classes, but so far no specific antibodies have been observed even after heavy immunization. Regarding the appearance of surface immunoglobulin positive (s-Ig+) cells, the most striking effect of bursectomy was observed in the frequency of s-IgG+ cells, which were markedly decreased in the spleen, peripheral blood, and thymus of the Bx birds when compared with the age-matched controls. s-IgM+ cells were significantly decreased in the peripheral blood and s-IgA+ cells in the spleen of the Bx birds. In contrast to these findings, s-Ig+ cells of all three classes were present in normal frequency in the bone marrow. Bursectomy had no effect on the total lymphocyte and other white cell counts in the peripheral blood. Likewise, no effect on the frequency of B-L (Ia-like) antigen-positive cells in different organs was observed.     

Thymic development in surgically bursectomized embryonic chicken: expression of PCNA, CD3, CD4 and CD8 markers

Little information is available on the functional relationship between bursa and thymus during chicken embryogenesis. We, therefore, investigated embryonic thymuses taken at 17 days in ovo from chickens bursectomized at 68-72 hours, with histological, histochemical (PAS, Alcian blue), and immunoreaction (anti-cytokeratin B, anti-PCNA/cyclin and anti-CD3, CD4 and CD8 antibodies) methods and compared these data with those from normal and sham-operated chickens of the same age. The bursectomized thymuses distinctly differed from normal and sham-operated thymuses: they were smaller, and the cortical zone was thinner and contained fewer epithelial cells and thymocytes. Only few cortical thymocytes were immunoreactive for PCNA, indicating low proliferative rate. More cortical thymocytes as compared with the normal, expressed CD3 on their cell membrane, whereas the thymocytes at the cortical-medullary border expressing anti- CD4 and anti-CD8 antidodies were less numerous than in normal thymus. The medullary zone contained few epithelial clusters made up of fewer cells than medullary clusters in normal chickens. Some cystic formations were enlarged and contained PAS- or Alcian-blue positive amorphous material. All these data suggest that early bursectomy affects both morphological and functional thymic development.     

Effect of bursectomy and thymectomy on cellular immunological reactions in chickens

The effect of thymectomy + irradiation and of bursectomy + irradiation on the rejection of skin grafts and injected homologous spleen cells was studied. Prolonged skin graft survival was found in thymectomized and irradiated chickens. Bursectomy together with irradiation and isolated irradiation did not affect the rejection of these grafts. Surgical operations together with irradiation had no effect on the rejection of an allogenein cellular graft. In one-day-old recipients, survival of the cells was prolonged in all the birds, while in 12-day-old recipients the cells were rejected at the normal time by both bursectomized and thymectomized birds. The possible explanations of these results are discussed. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

Ascaridia galli: effects of immunosuppressive drugs or bursectomy in chickens

Immunosuppressive drugs significantly increasing numbers of A. galli and incidences of infection were: cortisone, cortisol, 9-α-fluorohydrocortisone, 2-methyl-9-α-fluorohydrocortisone, prednisone, prednisolone, 6-mercaptopurine, 2, 6-diaminopurine, 6-thioguanine, 5-bromodeoxyridine, 5-fluorouracil, methotrexate, chlorambucil, and actinomycin D. These drugs and/or worm burdens significantly suppressed weight gains of hosts, and neither altered the male:female ratio of worms nor their growth. The following drugs neither altered worm burdens nor increased incidences of infection: corticosterone, 2-azaadenine, 8-azaguanine, azathioprine, 6-azauridine, busulfan, thio-TEPA, triethylenemelamine, vincristine, acriflavine, reserpine, and l-phenylalanine.Worm burdens and incidences of infection were increased significantly in chickens surgically bursectomized when 3 or 14 but not 35 days old. Chicks bursectomized in ovo with testerosterone propionate on Day 5 or 14 of incubation and infected on Day 14 after hatching developed significantly increased worm burdens and incidences of infection.Applying the Kolmogorov-Smirnov test for goodness of fit to data on increased worm burdens showed that the immunosuppressive drugs or bursectomy had a normalization effect on the statistical distribution.     

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. I. Immunocytochemical analysis of such chimeras by using species-specific monoclonal antibodies

Chick embryos were bursectomized at 5 days of incubation according to a novel surgical technique described in this article. This method yields birds that are able to hatch and are devoid of the physiologic deficiencies resulting from the previously used method, which involved resection of the cloacal and posterior embryonic region. The bursectomized embryos were grafted in situ with a quail bursa of the same age, which thereafter became chimeric through chick host hemopoietic cell invasion. By means of species-specific antibodies, the chimeric condition revealed 1) that the bursal epithelium expresses a unique antigenic determinant (MB1 determinant), until now considered to be an exclusive feature of blood vessel endothelium and hemopoietic cells, and 2) that this determinant appears in bursal epithelium at the time and site of hemopoietic cell invasion. The other point arising from this work concerns the apparent constitutive Ia expression by perifollicular blood capillary endothelial cells in normal and chimeric bursas.     

Evidence for the presence of precursor B cells in normal and in hormonally bursectomized chick embryos

Embryonic bone marrow of normal and hormonally bursectomized chicks was examined for the presence of hematopoietic precursor cells capable of migrating to the thymus and bursa and of differentiating into functional T and B cells, respectively. Following transfer of chromosomally marked bone marrow of normal and in ovo bursectomized 14-day-old embryos to 14-day-old γ-irradiated embryonic recipients, donor cells proliferated in the marrow, thymus, and bursa of recipients, and differentiated to PHA- and Con A-responsive T cells as well as to dextran sulfate- and anti-immunoglobulin-responsive B cells. In contrast, when marrow of 2-day-old hatched normal and in ovo-bursectomized donors was transferred to 14-day-old embryonic recipients, donor cells repopulated only the marrow and thymus of recipients which was followed by differentiation to Con A- or PHA-responsive T cells, but the same donor cells failed to proliferate in the bursa and there was no differentiation to functional B cells of donor type. The data were fitted to a model of T- and B-cell differentiation from the stem cell level and they suggest the presence of separate populations of committed precursor T (PT) and precursor B (PB) cells in the marrow of normal and in ovo bursectomized embryos with a bursa-independent selective disappearance of PB cells from the marrow during the late embryonic period.     

Effect of ACTH injection and graft of bursa of Fabricius on the adrenocortical activity of bursectomized chicks at 80 hours of incubation

Early embryonic bursectomy (BFX) disturbed the adrenocortical functioning. The stress-unresponsive period that occurred in controls, and lasted for 2-3 weeks after hatching, no longer appeared in BFX chicks. In contrast, the magnitude of the stress-induced hypercorticosteronemia was much lower in BFX than in sham-operated 5 week-old chicken. It was assumed that such adrenocortical dysfunction was due to bursal deprivation, since grafting bursal buds onto the chorio-allantoic membrane of BFX embryos restored all the parameters under study, i.e., the post-hatching stress unresponsive period and the high magnitude of stress-induced responses in adults. Factor(s) involved in such interregulation are not known but do not seem to affect directly adrenocortical cells because intramuscular injection of a moderate dose of ACTH resulted in the same hypercorticosteronemia whether 3 day-old and 5 week-old chicks had been bursectomized or sham-operated.     

Bursectomy in ovo blocks the generation of immunoglobulin diversity.

Immunoglobulins made by chickens bursectomized in ovo on day 11 of incubation were studied by two-dimensional gel electrophoresis. All such bursectomized chickens have limited diversity in their immunoglobulin molecules. A range of different degrees of diversity restriction was found in individual bursectomized chickens. The limited immunoglobulin diversity was stable for at least 20 weeks in that the same spots were found over this time span. Bursectomized chickens that responded to repeated antigen challenge had sheep erythrocyte- and dinitrophenol-specific antibodies of limited diversity. Different bursectomized responders made almost identical antigen-specific antibodies. The results were interpreted as indicating that individual bursectomized birds had been blocked at different points during the developmental generation of immunoglobulin diversity, and that the genetic mechanisms that underlie the developmental diversification of clones of B cells lead to a sequential generation of very similar antibody molecules in different embryos.     

The effect of bursectomy and thymectomy on the course of avian influenza virus infection

The effect of hormonal bursectomy and neonatal surgical thymectomy on the course of an avian influenza virus infection in chickens was studied. Analysis of the immunologic status of the chickens prior to infection included assay of natural agglutinins to rabbit red blood cells and induced agglutinins to sheep red blood cells, serum immunoelectrophoresis patterns, and in vitro effects of phytohemagglutinin on lymphocyte transformation. At 6 wk of age the chickens received the influenza virus intratracheally. Daily temperatures and mortality were recorded and HAI antibody titers were measured 7 and 14 days later. Completely thymectomized chickens were characterized by a failure of lymphocyte transformation to take place in two successive studies and absence of thymic remnants at autopsy. Bursectomy was associated with a significantly higher occurrence of temperature elevation (P < 0.05) and mortality (P < 0.001). Thymectomy had no significant effect on the course of the virus infection. Preliminary findings with passive administration of serum from immune animals also suggested a role for antibody in host resistance. These studies suggest cell-mediated immunity is less important than humoral immunity in recovery from avian influenza virus infection.     

Bursectomy of chicken embryos at 60 hours of incubation leads to an oligoclonal B cell compartment and restricted Ig diversity

Chickens that have been surgically bursectomized at 60 h of embryonic development usually generate Ig producing B cells; however, the bursectomized chickens are incapable of specific antibody responses, even after repeated immunization. In the present work, we analyzed the molecular basis of this immunodeficiency. In the bursectomized chickens, DNA sequencing revealed a repertoire of Ig L and H chains with a low number of different V-J and V-D-J joints, indicating an oligoclonal B cell compartment. In addition, the L and H chains belonging to each B cell clone had similar gene conversion events in the V region. In situ hybridization to Harderian gland tissue sections showed, that B cells of the bursectomized chickens were, however, capable of terminal plasma cell maturation. Thus, in chickens that were lacking the bursal microenvironment, 1) only a few B cell precursors differentiated into mature Ig-producing B cells, 2) low rate of gene conversion resulted in restricted Ig diversity. Regarding the chicken B cell differentiation, the present data support a model that the induction of B cell differentiation is a bursa-independent event, whereas the bursa of Fabricius has a crucial role in the amplification and diversification of the embryonic B cell repertoire.     

Cytochemical examination of peripheral blood lymphocytes in bursectomized chickens

The starting material were 86 day-old utility hybrid Tetra SL chicks. Beginning from their third week of life, 59 chickens were kept in two separate rooms "A" and "B" for 42-56 days. The values of temperature and cooling were somewhat different in rooms "A" and "B", however essentially not deviating from the accepted zoohygienic norms. The obtained results revealed a significant reduction of the activity of acid phosphatase in blood lymphocytes of 8-10 week-old chickens bursectomized in the neonatal period and kept in room "A". No such changes were found concerning ATP-ase and 5'-nucleotidase. Similar effect appeared in the lymphocytes of non-bursectomized chickens kept in room "B". Antigen stimulation (SRBC) of bursectomized and non-bursectomized chickens brought about an increased activity of all the three enzymes in blood lymphocytes. At the same time it should be emphasized that the increased activity of the enzymes tested was modulated by bursectomy and conditions of the medium.     

Ultrastructural studies on erythropoiesis in the avian thymus

The cortex of enlarging thymic lobes from adult haemorrhaged Quelea quelea were found to be similar to those of wild birds where the thymic enlargement was occurring naturally. A detailed stereological analysis of cells broadly designated as lymphoid, and the construction of models to account for the results, indicates that the enlarging thymic lobe contains both large and small blast cells, a heterogenous group of medium lymphocytes, erythroid cells, and two types of very small lymphocytes. The distinction between early erythroid cells and some lymphocytes, despite this detailed analysis is very difficult, but it is possible in enlarging thymic lobes that up to 42% of the lymphoid cells may have erythroid characteristics.     

Persistence of abnormal melanocytes in immunosuppressed chickens of the autoimmune “DAM” line

Summary The delayed amelanotic (DAM) line of chickens (Gallus gallus) is characterized by the postnatal elimination of melanocytes from regenerating feathers and from the choroid. The process of elimination is accompanied by a massive infiltration of mononuclear leukocytes (MNL) into the supporting connective tissues. When surgically bursectomized at day of hatching, chickens from this lineage develop significantly less amelanosis of the feathers. We report here a histological analysis of regenerating feathers and choroids from bursectomized birds that maintained their plumage pigmentation. In the feathers we observed the presence of morphologically abnormal melanocytes in the absence of MNL infiltration. Choroids also contained abnormal melanocytes without MNL infiltration; however, we observed a few cases of amelanotic choroids with a few MNL. These findings indicate that melanocytes of pigmented birds are morphologically abnormal even in the absence of a bursa and in the absence of leukocytic infiltrates into regenerating feathers and possibly into the choroids. We conclude from these findings that the amelanosis in unbursectomized DAM birds is due to the response of the immune system to an abnormality in the melanocytes which, by itself, does not lead to depigmentation.     

The presence of immunoregulatory cells in chicken thymus: function in B and T cell responses.

The presence of immunoregulatory cells in chicken thymus was studied by using several different systems. Chickens injected with large numbers of syngeneic thymocytes were tested for their ability to produce antibody to heterologous red cells. Similar chickens were studied for their ability to reject allogeneic skin grafts. In separate studies, mixtures of thymocytes with spleen cells or with peripheral blood leukocytes were assayed for their ability to respond to PHA or to produce a graft-vs-host reaction in embryonic chicks. These studies indicated that immunoregulatory cells exist in chicken thymus, which displays both helper and suppressor activity. The suppressor cells were more prevalent or more easily detectable in young birds and in chickens with intact bursas. The helper function of thymocytes was seen to better advantage with cells derived from older animals and from bursectomized donors.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

蛇胸腺胚胎发育的组织学研究

该文应用光镜、电镜和细胞计数技术对胚胎发育期虎斑颈槽蛇胸腺的发育分化进行了研究。在胚胎发育１１期,胸腺原基内出现前淋巴细胞。从胚胎发育１２期至出生前（１６期）,淋巴细胞不断增殖分化,小淋巴细胞逐渐增多,而淋巴母细胞和中淋巴细胞逐渐减少。胸腺皮质和髓质形成于１６期。巨噬细胞以及肌样细胞和胸腺ＡＰＵＤ细胞分别形成于胚胎发育１４期和１５期,随后数量有所增加,分别分布于胸腺皮质和髓质。     

Lymphokines in sensitized rats. III. Influence of prednisolone treatment of lymph node and spleen architecture in sensitized rats and upon MIF release in vitro.

The influence of prednisolone (corticosteroid, C.S.) treatment upon established cell-mediated immunity (CMI) induced by complete Freund's adjuvant (CFA) has been studied in rats by using in vitro migration inhibitory factor (MIF) assays, type IV skin reactions, and regional lymph node and spleen histology. Additionally, changes in the mononuclear-polymorphonuclear ratio of peripheral blood and T-cell accumulation in bone marrow in response to C.S. treatment have been determined. These results have been evaluated by comparison with equivalent experiments upon animals treated with anti-rat lymphocyte serum (ALS), oxisuran, or 2-[(methylsulfinyl)-acetyl]pyridine, which selectively suppresses CMI. The results suggest the existence of a population of “educated” T-cells in the thymic cortex of sensitized rats, and they suggest that prolonged C.S. administration does not suppress T-effector cells involved in established CMI but, rather, affects lymphocyte and monocyte migration patterns, including the migration of educated T-cells from the thymic cortex into other tissue compartments.     

A reexamination of humoral tolerance in chickens congenitally infected with an avian leukosis virus.

In a reexamination of congenital infection of chickens with ALV, one of the classical models of immunologic tolerance, we were unable to demonstrate that there was any evidence suggesting humoral immune reactivity to the infecting virus. In ALV-F42 congenitally infected birds that have a persistent viremia and no neutralizing antibody detectable by conventional means, we could find no evidence of host IgG deposits in the kidneys, nor any suggestion of renal pathology, or damage. In addition, attempts to precipitate any putative circulating infections virus-antibody complexes by treatment with a highly specific anti-globulin were negative nor were there titer differences in the viremic plasmas of bursectomized birds compared to nonbursectomized birds, indicating that the chickens were not making either a masked neutralizing or a nonneutralizing antibody response to the infecting virus. The evidence presented indicates that congenital infection of chickens with ALV does, at least at the humoral level, result in a state of immunologic tolerance to the infecting agent.     

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.