Studies on antilymphocyte antibody in the chicken. I. Effect of rabbit antithymus and antibursa globulin on chicken embryos

Antithymus globulin (ATG) and antibursa globulin (ABG) prepared in rabbits with thymus and bursa cells were used in experiments on New Hampshire embryos. The in vitro assay of potency of ATG and ABG by means of absorption, leucoagglutination, cytotoxicity test and passive haemagglutination showed that ATG and ABG react equally well with both thymus and bursa antigens.

Seven-day-old embryos were treated with eleven intrachorioallantoic injections of ATG, ABG and NRG (normal rabbit globulin) from the seventh to seventeenth day of incubation. The incidence of death in embryos receiving ATG and ABG was very high when compared with controls injected with NRG and saline. The ATG affected the cellular make-up of the spleen and bursa, while ABG influenced only the bursa. The cytotoxic-like and development-retarding activity of ATG and ABG were possible only in the presence of guinea-pig complement. The embryonic thymus proved to be resistant to the action of heterologous antilymphocyte antibody.

     

The influence of bursocytes and thymocytes on graft versus host reaction and IgG level in chickens.

Graft versus host reaction was induced by the blood of chickens previously transferred by the bursa and thymus cells. These bursa and thymus cells were taken from bursectomized or thymectomized chicken. It was occurred that graft versus host reaction was influenced by transfer of bursa and thymus cell. IgG level was affected by the transfer of these cells too.     

Antigenic surface determinants of chicken lymphoid cells. I. Serologic properties of anti-bursa and anti-thymus sera

The serologic properties of turkey antisera to chicken bursa (ABS) and thymus (ATS) cells were assayed by cytolysis-in-agar, conventional cytotoxicity tests and indirect immunofluorescence. Using proper absorptions the following antigenic surface determinants were detected on bursal and thymic lymphoid cells: (a) common lymphocyte antigens present on both kinds of cells; (b) thymus-specific antigens; (c) bursa-specific antigens; (d) in addition to the latter, bursa cells displayed immunoglobulin surface determinants. Even before absorption the anti-thymus and anti-bursa sera gave higher titres of reactions with homologous target cell preparations. In complement dependent cytotoxicity tests ABS and ATS reacted specifically with bursa and thymus cells respectively. In the spleen of 2- and 3-week-old chickens 20–30% of lymphoid cells were killed by ABS and 40–50% by ATS. The advantage of avian antilymphocyte sera for in vivo studies in chickens are emphasized.     

Characterization of avian lymphocyte surface proteins which bind to membrane and circulating immunoglobulins

Lactoperoxidase-catalyzed cell surface radioiodination was employed to label membrane proteins of chicken cells from bursa, and spleen cells from normal or bursectomized (Aγ) chickens. Detergent lysates of labelled cells were precipitated with rabbit anti-chicken Ig Fab antisera to isolate membrane immunoglobulins and with chicken anti-human gamma globulin: human gamma globulin immune complexes or insoluble, heat-aggregated chicken Ig to isolate Fc receptors (FcR). Resolution of bursa or normal spleen cell membrane proteins bound by anti-Fab antibodies by SDS-PAGE under reducing conditions revealed three molecular species (70,000, 45,000 and 25,000 dallons) specifically precipitated. All membrane proteins bound by the anti-Fab reagents were bound by antisera specific for chicken IgM. Bursa lymphocyte membrane proteins which bound to immune complexes were resolved into two major molecular species (55,000 and 45,000 daltons). The 45,000-dalton FcR exhibited an isoeleclric point and detergent sensitivily idenlical to a 45,000-dalton protein coprecipitated with membrane Ig. Two-dimensional gel analysis of Fc receptors obtained from spleen cells of bursectomized birds revealed that these proteins can be distinguished by size and/or charge from bursa Fc receptors. The results suggest that chicken lymphocyte Fc receptors comprise a group of proteins which can be distinguished by size, charge, and cellular origin.     

Tolerance to class I major histocompatibility complex antigens in chicken B cell chimeras. Effect of B cell depletion on transferability of tolerance

B cells from bursa of Fabricius of newly hatched chickens are able to reconstitute the B cell compartment of chemically bursectomized chickens. The resulting B cell chimerism can be detected with monoclonal antibodies against donor B cell alloantigen. Chimeric chickens accept donor-type skin grafts and are unresponsive to donor major histocompatibility complex (MHC) antigens in graft-vs.-host splenomegaly assay and mixed lymphocyte reaction. To study the capability of B cells to induce tolerance to selected MHC antigens, we transplanted class I or total MHC-incompatible bursa cells into cyclophosphamide-treated recipients. The recipients of class I or total MHC-incompatible bursa cells were equally tolerant of donor-MHC antigens. To further analyze the mechanisms of tolerance to class I antigens vs. total MHC, spleen cells from tolerant chickens were transferred to irradiated, histocompatible secondary hosts. The secondary recipients were also unresponsive to bursa cell donor-strain MHC antigens. However, if the chimeric B cells were depleted before the spleen cell transfer, the transfer of tolerance to total MHC was severely inhibited. Instead, most recipients of B cell-depleted spleen cells tolerant of class I antigens were still tolerant of bursa cell donor MHC. Our results indicate differences in the transferability of tolerance to class I antigens vs. entire MHC, although in primary recipients of bursa cells the tolerance is similar. These data suggest that a mechanism that is not dependent on the presence of donor cell chimerism contributes to the maintenance of tolerance to donor class I antigens. The transfer of tolerance to total MHC disparity requires the presence of chimeric cells indicating that donor alloantigen expression is needed for induction of tolerance in the secondary hosts.     

The role of the thymus for maturation of transferred bursa cells into immunocompetent B cells in chickens treated with cyclophosphamide.

Chickens injected with cyclophosphamide and X-ray irradiated in the newly hatched period were immunized with a mixture of sheep red blood cells, Brucella abortus and Salmonella pullorum at 4, 5 and 6 weeks of age, and were examined for serum antibody titres, serum immunoglobulin concentration and bursal and splenic structures at 7 weeks of age. The neonatal treatments suppressed completely or almost completely antibody responses, immunoglobulin production and formation of bursal follicles and splenic germinal centres. The transplantation of bursa cells into the chickens immunologically impaired by the treatments restored these functions and structures. In contrast, the transfer of bursa cells into chickens thymectomized, cyclophosphamide-treated and X-ray irradiated did not result in efficient restoration of the bursa-dependent immune system; 10-day-old bursa cells hardly restore the system, although 4-week-old bursa cells did so slightly. The chickens thymectomized, cyclophosphamide-treated, X-ray irradiated and repopulated with 10-day-old bursa cells were examined for the existence of functional B cells with the use of a syngeneic cell transfer system. The experiments verified that immunocompetent B cells had not developed in the chickens thus treated.     

Influence of antigenic stimulation on lymphoid cell traffic in the chicken

The influence of one intravenous injection of human serum albumin (HSA) on the pattern of cell migration from the bursa of Fabricius was studied in six-week old chickens. The bursa was labeled in situ with ['Hlthymidine 24 h after either HSA o r saline administration. Forty-eight hours following local labeling of the bursa, the chickens were killed and the transport of label to other lymphoid organs studied with a radiochemical method, based on measurements of tritium in DNA of the different organs, and with autoradiography. The radiochemical method revealed a significantly increased proportion of bursa-derived label in the spleen of the immunized chickens. This was associated with an increased number of heavily labeled bursa-derived cells in the autoradiograms from the same organ. The majority of the bursa emigrant cells in the spleen were localized in the red pulp, particularly in clusters of pyroninophilic cells. Their topographical distribution was not apparently influenced by the immunization. It is suggested that the increased homing of bursa-derived cells to the spleen may reflect a recruitment of antibody-forming cell precursors.     

Suppressor cells for antibody production in vivo, induced by bursa cell injection into agammaglobulinemic chickens, belong to A CT4-, CT8+, TCRI- subset of T cells.

The suppressor cells, induced in agammaglobulinemic chickens by injection of bursa cells and capable of inhibiting adoptive antibody production upon cotransfer with bursa cells, were characterized with respect to phenotype. They were found to be CT8+, CT4-, and TCRI- (gamma delta). No evidence could be obtained that histamine type 2 receptor bearing cells were involved in the suppression of antibody formation in vivo.     

The augmentation of antibody responses by preliminary intrabursal priming in the chicken.

Direct injection of antigen into bursal tissue of young chickens followed by subsequent intravenous immunization markedly stimulated agglutinin production against Brucella abortus. In contrast, preliminary intravenous immunization did not produce stimulation. The promoting effect of intrabursal injection was antigen-specific. Antigen injection into the bursa reduced the extent to which subsequent bursectomy suppresses the immune response. Bursa cells from young chickens which had been injected with antigen intrabursally were active in transferring the ability to give a secondary response to B. abortus when injected into bursectomized-irradiated chickens. The cells derived from chickens primed intravenously or from normal chickens were inactive. The implant of bursa cells from the 18-day-old chickens which had been injected with antigen intrabursally or intravenously at 11 days of age showed a promoting effect in restoring the ability to give secondary responses to both of B. abortus and Salmonella pullorum as compared with that of the implant of the bursa cells from unimmunized donors. These findings are discussed in relation to the existence of precursor cells that can respond to the antigen with a potential to enhance the antibody response to subsequent antigenic stimuli but are not yet mature enough to produce the antibody.     

Characterization of the immunosuppression accompanying virus-induced avian osteopetrosis.

Infection of chickens with a myeloblastosis-associated virus which induced a high incidence of osteopetrosis was accompanied by immunosuppression. The immunosuppression was manifested in the following ways. The weight of the bursa, spleen, and thymus was depressed in infected chickens. Infected animals had a diminished capacity to form hemolytic plaques in a direct assay. Spleen cells from osteopetrotic animals did not respond to phytohemagglutinin, and the spleen and bursa had a decreased proportion of cells possessing surface immunoglobulin. Osteopetrotic animals failed to show an age-dependent increase in the proportion of cells demonstrating surface immunoglobulin that was observed in normal animals. However, several individual chickens with heavy osteopetrosis responded to antigenic stimulation in a normal fashion, indicating that although immunosuppression usually accompanies avian osteopetrosis, it may not contribute directly to abnormal bone proliferation.     

Pathological changes in broiler chickens fed ochratoxin A and inoculated with Escherichia coli.

A study was conducted to evaluate the effects of ochratoxin A (OA) on Escherichia coli-challenged broiler chickens. One hundred and eighty-four one-day-old broiler chicks were divided into two groups of 92 chicks each, with one group fed a control mash diet and the other fed a mash diet containing 2 parts/10(6) OA. On day 14, each group was further subdivided into two groups with one group inoculated with E. coli O78 (1 x 10(7) colony-forming units/0.5 ml) by the intraperitoneal route, whereas the other group was not inoculated with E. coli. Four birds from each group were sacrificed at 1, 2, 3, 5, 7, 10, 14 and 21 days post-inoculation to record pathological changes in the liver, kidneys, heart, lungs, bursa, spleen and thymus. E. coli infection induced perihepatitis and pericarditis in the liver and heart, respectively, in chickens infected with E. coli alone or in OA-fed birds from 1 day post-infection (DPI) onwards. At 1 DPI, a thin fibrin layer covered the liver and heart; however, at subsequent days, the layer became thicker. E. coli infection did not produce appreciable changes in the kidneys, bursa or thymus. However, there was congestion of the lungs along with mononuclear cell infiltration. Ochratoxin feeding induced changes from 10 DPI onwards in chicks fed OA alone and those infected with E. coli. The changes in kidneys included swollen proximal convoluted tubules, degeneration of tubular epithelium and interstitial nephritis. Degenerative changes and mononuclear cell infiltration were recorded in the liver. There was atrophy of the lymphoid organs along with depletion of lymphocytes. Gross and histopathological changes were more severe in chickens fed OA and inoculated with E. coli than the chickens fed OA alone or those infected with E. coli, indicating combined action of these two.     

Marek''s disease in chickens: development of viral antigen in feather follicles and of circulating antibodies.

The infection of young chickens with Marek's disease herpesvirus (MDHV) leads to the early appearance of viral antigen in the bursa Fabricii, in the kidney tubular epithelium, and particularly in the epithelial cells of growing feather follicles (Calnek and Hitchner, 1969; Purchase, 1970). Viral antigen may persist in feather follicles over several months, whereas in neural lesions or in lymphoid tumors induced by MDHV viral antigen is either lacking or present only in a few cells (Calnek and Hitchner, 1969; Spencer and Calnek, 1970; Purchase, 1970). Haider et al. (1976) have pointed out the high diagnostic value of the double-diffusion agar-gel participitation test (Chubb and Churchill, 1968) using growing feathers of MDHV-infected chickens as antigen.     

Effects of fowl adenovirus infection on the immune system of chickens.

A virulent strain of serotype 8 fowl adenovirus (FAV) was isolated from an outbreak of inclusion body hepatitis (IBH) in broiler flocks. Post-mortem changes included characteristic liver lesions with intranuclear inclusion bodies in the hepatocytes and severe lymphocytic depletion in the bursa, thymus and spleen. The packed cell volume was reduced by 50 per cent or more and varying amounts of cell depletion were observed in the bone marrow. Typical IBH was reproduced in specific pathogen-free chickens inoculated orally with the FAV isolated from the natural infection. There was severe depletion of lymphocytes in the bursa, thymus and spleen of the experimentally infected birds and FAV antigens were detected by ELISA and immunocytochemical staining in various lymphoid tissues. Humoral antibody responses against sheep red blood cells, detected by the haemagglutination test, were decreased in the chickens infected with FAV. These findings suggest that the damage caused by replication of this virulent strain of FAV in lymphoid tissues compromises the immunological capabilities of infected chickens.     

Infectious bursal disease: distribution and persistence of the virus in inoculated chickens. Study on the transmission of the disease

The distribution, concentration and persistence of infectious bursal disease virus (IBDV) in the lymphoid organs of inoculated chickens, and its persistence in contaminated premises was examined. The virus only multiplied in the bursa of Fabricius where it induced degeneration and necrosis of the lymphoid cells. It persisted for 10 days in this organ and the highest viral concentrations were observed between the 4th and 8th day following inoculation. The virus was found at a low concentration in the spleen and thymus only during the viraemia phase. The inoculated chickens shed virus in the excreta during the first days of infection. The disease was transmitted to other chickens by direct contact with birds which had been inoculated 4, 10 and 14 days previously with IBDV. Litter on which infected chickens had been reared had a high level of infectivity for 30 days after removal from the chickens and still had some infectivity after 60 days. The long life of the virus in an infected house explains its persistence on infected farms and its transmission to successive flocks.     

Bursal and postbursal stem cells in chicken. Functional characteristics

Cyclophosphamide (CY)-treated or surgically bursectomized, CY-treated 3-day old chicks were injected with bursa or bone marrow cells from donors of different ages. Cell recipients and donors were isogeneic at the major histocompatibility locus. Antibody responses to sheep red blood cells and Brucella abortus, and microscopic morphology of spleen and bursa were assessed 5–6 weeks after the cell transplantation. Relative weight of the bursa was found to be a reliable indicator of the restoration of bursal structure. The results indicate that stem cells or progenitor cells for the B cell line in chicken can be divided into a bursal stem cell and a postbursal stem cell. Both of these cell types are effective in restoring antibody formation of CY-treated chickens. Bursal stem cells restore the bursal morphology; they are not capable of further maturation without the influence of the bursal microenvironment. This influence is not effected by bursa in a cell impermeable diffusion chamber; actual contact with the bursal stroma is necessary. Bursal stem cells are also capable of restoring the formation of germinal centers in the spleen of CY-treated chickens. Postbursal stem cells do not restore bursal structure and they do not need the bursal micro-environment for further maturation. They also have no clear effect on the formation of germinal centers in the spleen. Bursal stem cells are found in the bursa during the first few weeks after hatching. Postbursal stem cells start to appear in the involuting bursa and at the same time, in the bone marrow also. They are found as the majority of cells in the bursa and bone marrow after at least the 10th week following hatching. Early postbursal stem cells have already passed the education given by the bursa, but have not yet totally lost their capacity to induce germinal centers in the spleen. They restore germinal center formation even in surgically bursectomized recipients, demonstrating that presence of bursal follicles is not necessary for the production of germinal centers. The findings are discussed to stress the significance of two equally important factors in the development of immunity: the lymphoid cells themselves and, at each stage ontogeny, the proper microenvironment for their further function and maturation.     

Allotype suppression in the chicken III. Analysis of the recovery from suppression by neonatally injected or maternal antibodies

Injection of M-1 (Cμ), G-1 (CμgM) heterozygous chickens on the day of hatch with anti-IgM-1 antiserum induced allotype suppression from which chickens recovered over a period of approximately 4 months. The suppression of the serum IgM-1 levels was matched by a decrease in the number of splenic and peripheral blood B cells bearing the relevant IgM-1 allotype, and a compensatory increase in the number of cells bearing the alternative nonsuppressed IgM-1 allotype. However, the proportion of IgM-1-bearing bursal cells was only marginally altered. The recovery from suppression was due to B cell recruitment and could be abrogated by bursectomy. Allotype suppression induced in ovo or maintained by repeated injections of anti-IgM-1 anti-serum resulted in chronic suppression and depletion of the relevant peripheral as well as bursal IgM-1-bearing cells. Antibody titers of the relevant allotype in partially suppressed chickens generally correlated with serum allotype levels without clonal restriction in antibody response of the suppressed allotype.     

Expression of perforin-granzyme pathway genes in the bursa of infectious bursal disease virus-infected chickens

Infectious bursal disease (IBD) is an economically important immunosuppressive disease of chickens. The IBD virus (IBDV) actively replicates in B cells and causes severe bursal damage. Generally, T cells are refractory to infection with IBDV but are known to promote virus clearance. However, the mechanisms of T cell mediated viral clearance are not well understood. In this study, we evaluated the molecular mechanisms of cytotoxic T cell responses in the pathogenesis of IBD in chickens. Infection of chickens with IBDV was accompanied by the infiltration of CD4⁺ and CD8⁺ T cells in the bursa. There was an upregulation in the gene expression of important cytolytic molecules; perforin (PFN), granzyme-A (Gzm-A), DNA repair and apoptotic proteins; high mobility proteins group (HMG) and poly (ADP-ribose) polymerase (PARP) in the bursa of Fabricius (BF) whereas expression of NK (natural killer) lysin was downregulated. Importantly, PFN producing CD4⁺ and CD8⁺ T cells were also detected in the bursa of IBDV-infected chickens by immunohistochemistry. The Th1 cytokines, IL-2 and IFN-γ expression was also strongly upregulated, suggesting the activation of T cells. The findings of this study highlight the mechanisms of IBD pathogenesis and the role of cytotoxic T cells in the clearance of virus-infected cells.     

Systemic Mycoplasma synoviae infection in broiler chickens.

Systemic Mycoplasma synoviae infection in 47-day-old broiler chickens with septicaemic lesions and increased carcass condemnation rate is reported. The clinical history included respiratory signs and an enlarged keel bursa. Condemnations at the processing plant were due to airsacculitis and keel bursitis. Involvement of several organs, including the keel bursa, liver, spleen, brain, choroid of the eye, nerves and skeletal muscle associated with vasculitis, and the isolation of M. synoviae from the liver and keel bursa are only occasionally seen in field cases. Random amplified polymorphic DNA analysis of the M. synoviae isolated from the broiler chickens in this study had a different pattern when compared with the reference M. synoviae strains, WVU-1853, MS-H and F10-2AS, and another M. synoviae isolated from broiler breeders from the same company, but had a similar DNA pattern to an M. synoviae isolated from broiler chickens and turkeys owned by the same company. This finding suggests a horizontally acquired infection rather than vertical transmission.     

Direct stimulation of lymphoid tissue of the chicken. I. Antibody-producing, antibody-transferring and plaque forming capacity of the thymus, spleen and bursa of Fabricius following intrathymic and intravenous injection of antigen

Bovine serum albumin (BSA) and human 0 red blood cells (0 RBC) were injected into six thymic lobes or into the vein of 8-week old normal and neonatally bursectomized New Hampshire chickens in order to study the antibody production. Thymus and spleen cells from donors immunized by the intrathymic or intravenous route with BSA were used in the transfer of antibody response to homologous bursaless nonimmunized recipients. The thymus, spleen and bursa of chickens immunized with 0 RBC were inspected for the presence of plaque-forming cells. Intrathymic injection of BSA induced a prompt appearance of 2-mercaptoethanol (ME)-sensitive anti-BSA antibodies in a number of chickens, and progressive increase of antibody titer. Bursectomized chickens showed very weak primary antibody response (only antibody of ME-sensitive type) following intrathymic or intravenous stimulation with BSA. The amount of ME-sensitive hemagglutinins increased 2 days after intrathymic or intravenous administration of 0 RBC, but there was no immediate rise of hemagglutinin titer in intrathymically injected birds. Intravenous injection of 0 RBC was superior to intrathymic injection in inducing ME-sensitive and ME-resist ant he magglutinins. Thymus cells from donors immunized intrathymically with BSA were capable of transferring antibody response to neonatally bursectomized recipients. The most effective were thymus cells from BSA-injected lobes. Thymus and spleen cells from intravenously immunized chickens exhibited a lower capacity for transferring the production of antibody. On the other hand, thymus and spleen cells from bursaless donors immunized intrathymically with BSA failed completely to induce antibody production in nonimmunized bursectomized recipients. Plaque-forming cells were found in the thymus, spleen and bursa of chickens immunized intrathymically or intravenously with human erythrocytes. The results were interpreted as further support for the concept that the thymus is actively involved in immune reactions, and that the chicken thymus shares properties of both primary and secondary lymphoid tissues.     

Experimental modification of the growth of the bursa of Fabricius in chickens revealed by scanning and transmission electron microscopy

The ultrastructural changes occurring in the bursa of Fabricius of 3 and 7 d old chickens after adriamycin treatment are described. Male Hubbard chickens 7 d old, were injected with a intraperitoneal injection of adriamycin (5 mg/kg/die). All chickens were killed 24 h after the last injection, and the bursa was rapidly excised and fixed immediately for scanning electron microscopy and for transmission electron microscopy. The bursa underwent atrophy and showed epithelium desquamation. After 6 d treatment the dysepithelization was evident. Consequently follicles were made up mostly with large polyedric cells.