Pathology of the thymus,spleen and bursa of Fabricius in zinc-deficient ducklings

The weight and growth index of bursa of Fabricius, thymus and spleen were significantly reduced (P < 0.05 or P < 0.01) in zinc (Zn)-deficient ducks (Zn 22.9 mglkg diet) when compared with normal ducks. The G0/Gl phase of the cell cycle of the bursa of Fabricius, thymus and spleen was much higher, and the S and G2+M phases lower in Zn-deficient ducks than in the controls. Histopathologically, there was lymphocyte degeneration and depletion of lymphoid organs, and the reticular cells of thymus were also degenerate or necrotic in the Zndeficient group. The results demonstrated that Zn deficiency seriously inhibited the growth of lymphoid organs and caused marked pathology in the lymphoid organs. The results also showed that the effect of Zn deficiency on the primary lymphoid organs occurred earlier than on the secondary lymphoid organs. The effect of Zn deficiency was greatest on the bursa of Fabricius, followed by the thymus, and then the spleen.     

Changes in lymphoid organs and blood lymphocytes induced by vitamin A deficiency and Newcastle disease virus infection in chickens

The effect of vitamin A deficiency in the presence or absence of Newcastle disease virus infection (NDV, La Sota strain) on weight of lymphoid organs and on the number and type of circulating white blood cells (WBC) was investigated in chickens. Day-old chickens with limited vitamin A reserves were fed purified diets containing either marginal (ad libitum) or adequate (pair-fed) levels of vitamin A and at 21–28 days of age; half the chickens in each group were infected with NDV. Vitamin A deficiency resulted only in significantly lower absolute and relative weights of bursa of Fabricius and after infection both weights of bursa and thymus were significantly lower. Relative weight of spleen was significantly higher after infection irrespective of vitamin A status. Liver weights were not affected by vitamin A status and/or NDV infection. Both vitamin A deficiency and NDV infection resulted in lymphopenia, while the lowest number of WBC were observed in vitamin A-deficient chickens during the acute phase of NDV (5 days after infection). Subsequent to lymphopenia due to NDV infection, a marked lymphocytosis was observed in controls and to a lesser extent in vitamin A-deficient birds. These results indicate that vitamin A deficiency, which is aggravated by concomitant NDV infection, affects lymphoid cell systems.     

Age-related changes and distribution of T cell markers (CD3 and CD4) and toll-like receptors(TLR2, TLR3,TLR4 and TLR7) in the duck lymphoid organs

T lymphocytes and Toll-like receptors have been confirmed to have correlation with the ability to resistance to pathogenic challenges and play an important role in duck immune system. However, the information of ontogeny of T lymphocytes and Toll-like receptors is scarcely in duck. Therefore, to address these questions, we report the development and distribution of CD3 and CD4 by immunocytochemistry and the age-related mRNA level of duck T cell markers (CD3 and CD4) and Toll-like receptors (TLR2, TLR3, TLR4 and TLR7) by real time quantitative PCR in duck lymphoid organs (thymus, bursa of Fabricius and spleen). Results indicated that CD3 and CD4 positive cells can be observed in all test organs and partly change in an age-related way. CD4 positive T cell of duck spleen mainly distributed in periarterial lymphatic sheaths and red pulp, not in white pulp. Both of CD3 and CD4 were experienced significant increased wave twice in duck lymphoid organs and T cell dependent cellular immunity of duck may well established until 5 weeks old. The mRNA expression levels of duck TLRs were age and organ dependent, and duck TLR3 and TLR7 were significantly lower abundance in the spleen but higher in thymus and bursa of Fabricius, respectively. This study provide the essential knowledge of the ontogeny of T cells and Toll-like receptors in duck, which may shed lights on the T-cell mediate immunity and innate immunity in duck.     

Studies on the chicken lymphocyte receptor for aggregated IGG

Chicken lymphocytes isolated from different lymphoid organs were analyzed for Fc IgG receptor by binding of heat aggregated IgG (agg IgG) in the indirect immunofluorescence test. The binding of agg IgG was shown to be specific for Fc IgG receptor, since IgM and F(ab′)₂ fractions were bound to the lymphocytes in a much less degree. The highest frequency of agg IgG binding cells, almost 100 %, was found in the bursa of Fabricius. In contrast to bursa, only a small percentage of thymus cells bound agg IgG. Dose-dependent experiments showed that binding of agg IgG to thymus cells takes place only at high concentrations of the aggregates whereas bursa cells bind agg IgG also at low concentrations. The finding indicates the existence of high and low avidity agg IgG receptors and/or different density of Fc receptors on bursa and thymus cells. A far lower percentage of EA rosette forming cells than of agg IgG binding cells in the various lymphoid organs may depend on the existence of two different Fc IgG receptors on the chicken lymphocytes.     

Comparative natural killer cell activities of thymic, bursal, splenic and intestinal intraepithelial lymphocytes of chickens

The natural killer (NK) cell activities of spleen, thymus, bursa, peripheral blood and gut intraepithelial lymphocytes (IEL) from FP and SC chickens were investigated in 4-hr and 16-hr 51Cr release assays. Target cells were 4 different tumor cell lines derived from either an avian leukosis tumor transplant (LSCC-RP9, LSCC-RP12) or from Marek's disease lymphomas (MDCC-MSB-1, MCDD-CU36). Great variability in cytotoxic potential was observed among NK cells of different lymphoid organs. NK cell cytotoxicity varied depending upon the type of effector cells, type of target cells, the ratio of effector to target cells, and the age and genetic background of chickens. Substantial levels of NK cell activity were detected in spleen and gut IEL of SC chickens in a 4-hr assay. In contrast, the NK cytotoxicity in gut IEL of FP chickens was not detectable until 16 hr after incubation. The ranges of target cell specificity demonstrated by IEL, spleen, thymus and bursa NK cells were similar to one another and, in general, the level of cytotoxicity increased with incubation time. Thymus and bursa NK cell activity of both SC and FP chickens was not detectable in a 4-hr assay but substantial NK cell activity was demonstrated in a 16-hr assay. The results of the present study demonstrate that various lymphoid organs of chickens, such as spleen, thymus, bursa, and gut intraepithelium, contain subpopulations of cells that can mediate spontaneous cytotoxicity.     

Solubilization and partial purification of lymphocyte specific antigens in the chicken

Four different lymphocyte antigens were solubilized from chickens homozygous at the B locus. The antigens were identified by immunoelectrophoresis with rabbit anti-lymphocyte sera. 1. A bursa-specific cell surface antigen was extracted with 3 M potassium chloride and partially purified. The antigen was excluded from Sephadex G-200 gels, was heat labile and was a potent immunogen. The antigen could be detected on all bursa cells by immunofluorescence but not on thymus, spleen or blood lymphocytes. The findings do not exclude, however, the possibility that a small number of lymphocytes (e.g. plasma cells) possess the antigen. 2. A thymus-specific antigen was obtained by papain treatment of thymus cells. It migrated cathodically in immunoelectrophoresis. 3. A surface antigen which was specific for lymphocytes from both central lymphoid organs (thymus and bursa) was solubilized by pestle homogenization. 4. A fourth lymphocyte surface antigen was present on lymphocytes from blood, spleen, bursa and thymus. It was best solubilized by 3 M potassium chloride extraction and did not migrate under the conditions of immunoelectrophoresis. Studies did not reveal a stable antigenic marker with specificity for thymus or bursa cells and their progeny in the peripheral lymphocyte pool.     

Immunological capacity of the chicken embryo. I. Relationship between the maturation of lymphoid tissues and the occurrence of cell-mediated immunity in the developing chicken embryo.

In an investigation of the ontogeny of lymphoid tissue in chick embryos to relate maturation of lymphocytes with immunological competence, the numbers and sizes of lymphocytes were determined in the thymus, bursa of Fabricius, spleen, femoral marrow and peripheral blood of embryos from the 12th to 21st day of incubation, and in 6-day-old chicks. Results showed the thymus to be the first fully developed and most active lymphocytopoietic organ, followed by the bursa. The bone marrow was not lymphocytopoietic; the spleen and bone marrow were mainly granulocytopoietic and erythropoietic; some morphological differences between thymic and bursal lymphocytes were shown by light microscopy. It appears that in embryos and young chicks the lymphocytes are derived from the thymus and bursa, but not the bone marrow. In tests of immunological competency, cells of the thymus, bursa, spleen, bone marrow and peripheral blood from 12--21-day-old embryos and 6-day-old chicks were transferred to chorioallantoic membranes of 12-day-old recipient embryos. There were distinct differences between the ability of various lymphoid tissues to induce formation of chorioallantoic pocks or splenic enlargement. The thymus, spleen and peripheral blood elicited both lymphocytic pocks and splenomegaly, the bursa elicited splenomegaly only, and the bone marrow was ineffective. The bone marrow, however, induced formation of nonlymphocytic pocks. It is concluded that the immunological activity of the chicken embryo is primarily effected by the thymus and bursa and that cell-mediated immunity appears in the 2nd week of incubation.     

Immunoglobulin-bearing lymphocytes of the chicken. I. Heavy chain immunoglobulin commitment and organ distribution

When purified anti-immunoglobulin light chain antibodies were used in indirect immunofluorescence or labeled with 125I for autoradiographic staining, a similar percentage of Ig-bearing lymphocytes were detected by both techniques in lymphoid cell suspensions from the thymus or blood of 8-14-week-old chickens. However, a larger proportion of Ig positive lymphocytes were detected in suspensions of bursal cells by the more sensitive autoradiographic method, suggesting a lower surface density of Ig: perhaps on newly differentiated stem cells. In thymus and spleen suspensions, the proportions of Ig positive lymphocytes carrying mu and gamma-chains were roughly equal, whereas in the B cell populations of the bursa and blood, cells carrying surface gamma-chains predominated. IgA-bearing lymphocytes were only a minor population (< 5%) in lymphocyte suspensions prepared from the thymus, bursa, blood and spleen of adult chickens, but formed almost 50% of the Ig-bearing lymphocytes in the caecal tonsils.     

Chicken anemia virus infection in broiler chickens in Shahrekord, Iran: serological, hematological, and histopathological findings

Chicken infectious anemia caused by a single-strand DNA circovirus is a disease in young chickens that is characterized by aplastic anemia, generalized lymphoid atrophy, and immunosuppression. In the present study, the presence of chicken anemia virus (CAV) infection and the hematologic and histopathologic changes in CAV seropositive broiler chickens in Shahrekord region, center of Iran, were investigated. Blood and lymphoid tissue samples were obtained from 271, 2–6-week-old chicks of 23 commercial broiler chicken flocks. Measurement of CAV antibody titers by enzyme-linked immunosorbent assay (ELISA) and differential leukocyte counts were carried out on serum and whole blood, respectively. The results of ELISA showed that all flocks of different ages were positive for anti CAV antibodies. Histological examination of lymphoid organs revealed lymphocytic depletion in 72 chicks, of which 66 cases were seropositive in ELISA. Twenty chicks had lymphocytic depletion in both thymus and bursa of Fabricius and 46 cases had only bursal lymphoid depletion. Macroscopic appearance of bone marrows and spleen were normal. The mean antibody titers, packed cell volumes (PCVs), and number of lymphocytes and heterophils of the chickens with both thymic and bursal lymphoid depletion were significantly different than antibody titers and PCVs of the other group. These results have demonstrated CAV infection and immunosuppressive effect of this virus in broiler chickens and the need for vaccination of breeder flocks in center of Iran.     

Expression of pituitary adenylate cyclase-activating polypeptide in the primary lymphoid organs of the duck Anas platyrhynchos

The expression of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the thymus and bursa of Fabricius of the duck Anas platyrhynchos, at different ages, using immunohistochemistry, Western blotting, RT-PCR and sequencing. In the thymus, PACAP immunoreactivity (-ir) was found in lymphoid cells. CD68/ and PGP 9.5/PACAP38 double labelling showed that PACAP was not expressed either in macrophages or in epithelial cells, suggesting that the PACAP-positive cells observed were lymphoid cells. Immunoreactive lymphocytes were observed in the interlobular septa. They increased in number with ageing. In the bursa, PACAP-ir was found in nerve fibres and in a few lymphoid cells. RT-PCR revealed PACAP mRNA expression in the thymus but not in the bursa. These results suggest that PACAP plays a role in the functions of the immune system in birds.     

Chemical bursectomy of chickens with colchicine applied to the anal lips.

To induce chemical bursectomy, 30 microliter colchicine dissolved in saline solution (1 mg/ml) was applied on the anal lips of White Leghorn chickens once daily for four consecutive days after hatching. Histologic characteristics of the bursa of Fabricius, spleen, thymus, cecal tonsils, and rectal wall were studied 1-7 days after hatching. Total necrosis of the lymphoid cells and the follicle-associated epithelium in the bursa was observed during the four days of colchicine application. The bursal stroma remained unchanged, and only minor changes were found in the interfollicular surface epithelium. After colchicine application ceased, some regeneration of the epithelium, as evidenced by small epithelial buds, was found. At the end of the observation period the epithelial buds were often covered by the follicle-associated epithelium, which was capable of phagocytizing carbon. However, practically no lymphoid repopulation was seen in the buds. Since this method of colchicine application had no direct effect on other lymphoid organs or on the survival or weight of the chickens, this bursectomy model seems to be a new tool for use in studies of bursal function.     

Ontogeny of the immuno-neuro-endocrine relationship. Changes in lymphoid tissues of chick embryos surgically decapitated at 33–38 hours of incubation

The prosencephalon, and primordium of the hypophysis were surgically removed from chick embryos at 33–38 hours of incubation. The thymus, bursa of Fabricius, spleen, bone marrow and liver were examined cytomor-phologically on day 15, 17 and 19. T marker-bearing and Bu marker-bearing lymphocytes were identified by immunofluorescence. Decapitated embryos tended to be smaller than sham-decapitated controls of the same age, and exhibited retarded development of the thymus, bursa, spleen and liver. Decapitation particularly affected the cellular composition of the bursa and spleen, induced a decrease in the number of lymphocytes, and caused a striking depletion of lymphocytes bearing Bu antigen. This experiment showed an interdependence between lymphoid (immune), nervous and endocrine centers in the chick embryo.     

Immunocytochemical localization of vitamin D-dependent calcium-binding protein in the growing chick lymphoid organs

Monospecific antiserum against chick duodenal vitamin D-dependent calcium-binding protein (D-CaBP) was used to localize this protein by the peroxidase-antiperoxidase method (PAP) in the thymus, spleen and bursa of Fabricius of normal growing chicks in 20 day old embryos; in normal growing chicks at 1, 2, 3, 4 and 6 weeks of age in chicks fed a rachitogenic diet for 4 weeks. In the normal chick thymus, D-CaBP was localized throughout the cytoplasm and in the nucleus of cortical epithelial reticular cells (ERC) and in Hassal's corpuscles of the medulla. In the normal spleen reticular cells of the marginal zones showed dense deposition of reaction product in the nucleus and throughout the cytoplasm. In the bursa of Fabricius, only a few scattered cells in the medulla showed some staining. Wide variation was encountered in D-CaBP staining in the thymus and spleen of 4 week old chicks from different broods. In 4 week old rachitic chicks, staining in the thymus and in the spleen was generally reduced in intensity and occasionally was entirely absent. The presence of D-CaBP in some reticular cells of the thymus, spleen and bursa of Fabricius, identifies these lymphoid organs as vitamin D3 targets. Thus, 1,25(OH)2D3 may have an important role in some aspects of the immune defence mechanism.     

Pathogenesis of infectious bursal disease in embryonally bursectomised chickens

Embryonally bursectomised and nonbursectomised chickens were infected with infectious bursal disease virus (IBDV) at 36 days of age. Neither clinical signs nor gross lesions were observed in the infected, bursectomised (IB) chickens. No significant changes were observed in carcass, thymus or spleen weights of IB and noninfected bursectomised chickens. A mild lymphocytic necrosis and depletion were found in the spleen, thymus and caecal tonsil of the IB chickens. Neither precipitating nor serum neutralising antibodies were detected in the sera but IBDV was reisolated from the spleen and thymus. Infected, nonbursectomised (IN) chickens developed severe depression with diarrhoea and high mortalities. Haemorrhages were found in the muscles of the breast and thigh, proventriculus and intestines. Significant changes were observed between the carcass, thymus and bursa weights of the IN and noninfected, nonbursectomised chickens. There was severe lymphatic necrosis of the bursa, thymus, spleen and caecal tonsil. Both precipitating and neutralising antibodies were detected in the sera and the virus was reisolated from the bursa, thymus and spleen. It is concluded that the bursa of Fabricius is not essential for the establishment of an IBDV infection but is required for the clinical infection.     

Histopathology of chicks inoculated with chicken anaemia agent (MSB1-TK5803 strain)

One-day-old chicks, inoculated intramuscularly with the MSB1-TK5803 strain of chicken anaemia agent (CAA), showed a decrease of haematocrit value and inhibition of body weight gain, particularly between days 12 and 20 post inoculation (pi). Macroscopically, yellowish bone marrow, atrophy of the thymus and bursa of Fabricius, enlargement and discoloration of liver were observed in most chicks at the peak of infection. Histologically, the lesions appeared first in bone marrow and thymus on day 6 pi, and then in the bursa, spleen and liver. Swelling and intranuclear inclusion bodies in the haematopoietic precursor cells, thymocytes and reticular cells of the thymus were considered to be characteristic lesions. Hypoplasia or aplasia of bone marrow, depletion of lymphocytes in the lymphoid organs and swelling of hepatocytes occurred during the anaemic phase. Repopulation of the haematopoietic cells and lymphocytes occurred between days 16 and 24 pi, and most tissues of the surviving chicks recovered by day 32 pi.     

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.     

Immunopathology of chickens infected in vivo and at hatching with the avian osteopetrosis virus MAV.2–0

The immunological capacity of chickens infected with MAV.2–0, an avian osteopetrosis virus, was studied both morphologically and functionally. Infection of 11 to 12-day-old embryos with a high (0.58 × 10⁶-1.2 × 10⁶ plaque-forming units; PFU) and an intermediate (5.8 × 10⁴ PFU) dose of virus resulted in severe stunting, as manifested by lower body and lymphoid organs (bursa, thymus and spleen) weights. The bursa and spleen of osteopetrotic chickens were poorly developed, the thymus partially necrotized; all lymphoid organs had fewer lymphocytes than controls, and there were far fewer germinal centers in the spleen of infected birds, as compared to controls. The humoral and cellular immunity of these osteopetrotic chickens was significantly suppressed, as assessed by (a) the plaque-forming cell response against sheep red blood cells (SRBC) in the spleen; (b) circulating antibody responses against SRBC, Brucella abortus and human γ-globulins (HGG); (c) the delayed hypersen-sitivity reaction against HGG; and (d) mitogenic responsiveness of peripheral blood and spleen lymphocytes to concanavalin A, phytohemagglutinin M and pokeweed mitogen. A few birds, infected as 11-day embryos with the lowest concentration of virus (2.9 × 10⁴ PFU) had no palpable bone lesions, the lymphoid organs had normal histology, and immune responses were quite similar to those in uninfected control birds. Osteopetrotic chickens, infected within 48 h after hatching (5.8 × 10⁵ PFU), had normal IgM-class antibody responses against all antigens studied (SRBC, Brucella, HGG), whereas IgG and/or IgA responses tended to be lower than those observed in the normal controls. These findings, together with the regularly organized small lymphoid follicles in the bursa, indicate a late affection of B cell development, whereas in the birds infected at 11–12 days of incubation, an almost total arrest of B cell development was observed. T cell functions of birds infected at hatching were suppressed to the same extent as those of in ovo infected birds indicating susceptibility of the T cell lineage to MAV.2–0 also at later stages of development.     

Immune globulin production by the bursa of Fabricius of young chickens

A study was made of the formation of immune globulins in vitro by lymphoid tissues from neonatal chicks. Incorporation of ¹⁴C-amino acid into γM and γG was demonstrated by means of radioimmunoelectrophoresis. Results were correlated with observations made on tissue sections stained for the presence of immune globulins with fluorescein-labelled antisera. The bursa of Fabricius synthesized γM as early as the 18th day of embryonic development, and was significantly more active than the spleen in the production of immune globulins during the 1st week after hatching. Medullary cells of the bursa follicles contained immune globulins. Synthesis of γM by the spleen became detectable on day 7. Comparable activities in immune globulin formation in bursa and spleen were obtained by day 15, while the spleen was much more active than the bursa in 3-month-old chickens. The thymus produced no γM but started to produce γG during the 2nd week after hatching. Bursa and spleen from 8-day-old germ-free chicks showed similar synthesis of γM, as did organs from normal animals. The significance of this early γM synthesis and its possible independence of antigenic stimulation are discussed.     

Studies on antilymphocyte antibody in the chicken. II. Effect of antithymus and rabbit antibursa globulin on immune reactions in young chickens

Four-week-old chickens repeatedly injected intraperitoneally with heterologous antithymus globulin (ATG) and antibursa globulin (ABG) were immunized, in the course of treatment, with bovine γ-globulin (BGG). The ATG and ABG depressed the production of anti-BGG antibody. On the other hand, only ATG was effective in suppressing experimental allergic encephalomyelitis. Immunoelectrophoretic analysis of sera from chickens treated with ATG, ABG and NRG (normal rabbit globulin) revealed that those globulins are themselves immunogenic. ATG and ABG induced in the spleen a moderate depletion of lymphocytes and plasma cells respectively. These reagents produced lymphocytopenia and granulocytopenia, but a similar, although less expressed, effect on leucocytes in the peripheral blood could be induced by NRG. Thymus, bursa, caecal tonsil, Peyer's patches and lymphoid masses of the intestine were not influenced by ATG, ABG and NRG. The results are discussed and interpreted as further evidence for the delineation between immune functions of the thymus and the bursa of Fabricius in the chicken.     

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.