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.     

Pathogenesis of infectious bursal disease in cyclophosphamide-treated chickens

Broiler chickens inoculated with cyctophosphamide showed atrophic bursae and severe immuno-suppression. Infectious bursal disease virus (IBDV) inoculation of the birds at 5 weeks of age caused neither clinical signs nor gross lesions. IBDV was re-isolated from some bursae samples. Cyclophosphamide non-treated (CYNT) chickens inoculated with IBDV showed bursal enlargement followed by atrophy. Examination of sections of the organ showed severe lymphocytic necrosis and depletion. IBDV was re-isolated from the bursa and spleen. The geometric mean titres of the IBDV serum neutralizing antibodies for infected cyclophosphamide treated (CYT) and CYNT chickens were 4.3 and 955.4, respectively. The above observations show that CYT chickens did not develop clinical IBD in the presence of infection and severe immunosuppression. They confirm the hypothesis that a healthy bursa is essential for the development of clinical IBD in chickens within the susceptible age range.     

Infectious bursal disease DNA vaccination conferring protection by delayed appearance and rapid clearance of invading viruses

The present study was undertaken to determine the kinetics of viral load and immune response in protection against infectious bursal disease virus (IBDV) by DNA vaccination. Chickens were DNA-vaccinated and challenged with IBDV one week after the third vaccination. Tissues were collected at 12 hours postinfection (HPI), 1 day postinfection (DPI), 3, 5, 7 and 10 DPI. The vaccinated chickens had less viral RNA, with delayed appearance and shorter duration in the bursa of Fabricius, spleen, and cecal tonsil than the challenged control chickens. Their ELISA and neutralizing antibody titers were decreased at 12 HPI and significantly lower (P < 0.05) than those in the challenged control chickens at later time points. Their spleen IFNγ expression was up-regulated compared to that in the DNA-vaccinated chickens without IBDV challenge. These results indicate that DNA vaccination confers protection against IBDV challenge by delayed appearance and rapid clearance of the invading viruses.     

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.     

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.     

The pathology of infection of chickens with the lentogenic V4 strain of Newcastle disease virus

Seven-week-old chickens infected oro-nasally with the lentogenic V4 strain of Newcastle disease virus showed no clinical signs and minimal gross pathology. There was slight ulcerative tracheobronchitis but the main system response was a rapid and progressive lymphoproliferative hyperplasia in the respiratory system, gastrointestinal tract, bursa and spleen which tended to peak after three weeks. By using an indirect immunoperoxidase technique with antibody prepared against homologous virus, viral antigen was localised chiefly in the cytoplasm of lymphoreticular cells, persisting for at least 28 days in the caecal tonsil. Positive cells were not seen in the bursa or brain.     

The working mechanism of an immune complex vaccine that protects chickens against infectious bursal disease.

The role of immune complexes (Icx) in B-cell memory formation and affinity maturation allow for their potential use as vaccines. Recently, a new immune complex vaccine has been developed that is currently under field trials conducted in commercial poultry. This immune complex vaccine is developed by mixing live intermediate plus infectious bursal disease virus (IBDV) with hyperimmune IBDV chicken serum (IBDV-Icx vaccine). Here we have investigated the infectivity of this vaccine as well as the native IBDV (uncomplexed) vaccine in terms of differences in target organs, in target cells and speed of virus replication. At various days after inoculation on day 18 of incubation (in ovo) with either one dose of virus alone or the IBDV-Icx vaccine, the replication of IBDV and the frequency of B cells and other leucocyte populations were examined in the bursa of Fabricius, spleen, and thymus using immunocytochemistry. With both vaccines, IBDV was detected associated with B cells, macrophages and follicular dendritic cells (FDC) in bursa and spleen, although complexing IBDV with specific antibodies caused a delay in virus detection of about 5 days. Most remarkable was the low level of depletion of bursal and splenic B cells in IBDV-Icx vaccinated chickens. Furthermore, in ovo inoculation with the IBDV-Icx vaccine induced more germinal centres in the spleen and larger amounts of IBDV were localized on both splenic and bursal FDC. From these results we hypothesize that the working mechanism of the IBDV-Icx vaccine is related to its specific cellular interaction with FDC in spleen and bursa.     

Pathogenicity of chicken anaemia virus (isolate 10343) for young and older chickens.

One-day-old chicks, inoculated intramuscularly (i.m.) with the chicken anaemia virus (CAV) isolate 10343, showed depression of body weight gain and anaemia, particularly between days 14 and 21 post-inoculation (p.i.)- The weights of thymus and bursa were substantially reduced compared to controls at days 14 and 21 p.i. The histological lesions detected in thymus, bursa, spleen and liver were similar in frequency at days 14 and 21 p.i. Eosinophilic intranuclear inclusion bodies, lymphocyte depletion, and focal necrosis were detected in the thymus, spleen, bursa and liver of more than 50% of the inoculated chicks at days 14 and 21 p.i. Focal necrosis and vacuolar degeneration in the liver, as well as apoptosis in different organs were more evident at days 14 and 21 p.i. Ten-week-old broiler breeders, inoculated i.m. with isolate 10343 showed pathological changes that were less severe than the changes shown by 1-day-old chicks. No anaemia could be detected in this group. However, severe thymus atrophy, and histological lesions in bursa, spleen, and liver, were also evident at days 14 and 21 p.i. in some of the inoculated birds. Viral detection by immunofluorescence using a monoclonal antibody revealed a wide distribution of the CAV isolate. CAV antigen was detected until day 21 p.i. in thymus, spleen, bursa and liver. According to the severity of the lesions shown by 1-day-old chicks, the length of the period in which CAV antigen could be detected in tissues, and the fact that CAV isolate 10343 was capable of inducing disease in 10-week-old chickens, it seems that this CAV isolate may be particularly virulent.     

Effect of infectious bursal disease virus on infections produced by Escherichia coli of high and low virulence in chickens

The effect of infectious bursal disease virus (IBDV) on the infections caused by Escherichia coli strains of high (Expt 1) and low (Expt 2) virulence was examined in specific-pathogen-free chickens. The chickens were inoculated orally with IBDV at 1 day of age and via the air sac with E. coli at 1 week of age. In the groups given 1 x 10(5) cfu of E.coli of high virulence (Expt 1), mortality of IBDV-inoculated group (90%) was significantly higher than that in the non-IBDV-inoculated group (40%). The septicaemic lesions (splenic necrosis with fibrinous exudation) in the IBDV-inoculated-group were of significantly greater severity than those in the non-IBDV-inoculated group. The lymphocytic depletion in the bursa of Fabricius was most severe in the group inoculated with both IBDV and E. coli, then in descending order, in the group inoculated with IBDV alone and with E. coli alone. Lymphocytic depletion of the thymus was caused mainly by E. coli infection while IBDV induced mild lymphocytic depletion of the thymus. In Expt 2. the groups given 1 x 10(9) cfu of E. coli of low virulence revealed mortality of 50% when inoculated with IBDV and 10% when non-IBDV-inoculated. This study suggests that IBDV may increase the chickens' susceptibility to septicaemic infections produced by E. coli strains of high and low virulence and that IBDV and E. coli may induce additively marked lymphocytic depletion in the bursa of Fabricius and thymus.     

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.     

The use of monoclonal antibody probes for the detection of infectious bursal disease virus antigens

Two monoclonal antibodies (MAb), 2E6 and 2G10, were used against infectious bursal disease virus (IBVD) P3009 in an immuno-dot assay to detect IBDV antigens from cell culture, and from bursa and spleen tissue samples of chickens. The limit of viral antigens detected by using both MAb probes was 48 ng. The probes were used to detect five serotype 1 IBDV isolates and one serotype 2 IBDV strain. The result indicated that these probes had broad specificity. The probes, however, did not cross-react with viral antigens prepared from seven unrelated avian viruses. The probes detected IBDV antigens in bursa and spleen tissues collected from chicks as early as 2 days after inoculation. The IBDV antigens in bursa tissue samples from six poultry farms were detected by both probes. However, tissue samples from two of six farms did not react with probe 2E6. The data suggest that it is possible to use two MAb probes for diagnosis of IBDV infection in poultry farms.     

Effect of infectious bursal disease on natural killer cell activity and mitogenic response of chicken lymphoid cells: role of adherent cells in cellular immune suppression.

A pathogenic isolate of infectious bursal disease virus (IBDV) caused persistent and extensive lesions in the bursa but mild and transient lesions in the thymuses of chickens of lines 63 and P. The effect of IBDV on two cellular immune functions, namely, natural killer cell cytotoxicity and mitogenic response, was studied. The natural killer cell activity was not consistently influenced, but the virus, during the first 2 weeks of infection, caused transient depression of the blastogenic response of spleen cells to phytohemagglutinin. Studies on mitogenic hyporesponsiveness revealed that the functional impairment was mediated by a suppressor cell that shared several characteristics with macrophages; i.e., the suppressor cell was adherent to plastic, was phagocytic, and resisted treatment with antithymocyte and antibursa cell sera. Removal of suppressor cells from the spleens of virus-infected chickens resulted in restoration of the mitogenic response of cells. Further, in mixing experiments, the suppressor cell isolated from the spleens of virus-infected chickens also inhibited the mitogenic response of normal spleen cells. We concluded that reduced mitogenic response of lymphocytes in IBDV-infected chickens was not due to a lack of functional T-cells, as suggested previously by others, but was due to macrophage-like suppressor cells. The suppressor cells, although present in certain normal chickens, became activated during early stages of IBDV infection.     

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.     

Detection of infectious bursal disease viruses by using cloned cDNA probes.

A molecular clone representing 445 base pairs at the 3' end of infectious bursal disease virus (IBDV) genome segment B was used in a dot blot hybridization assay to detect viral RNA from cell culture and from chicken bursa and spleen tissue specimens. The cloned nucleotide sequence represents approximately 14% of the virus-encoded polymerase (VP-1) gene. The lower detection limit of radiolabeled probes prepared from this clone was 0.1 ng of IBDV double-stranded RNA. The probe had broad specificity and was used to detect four serotype 1 IBDV strains and one serotype 2 IBDV strain. This probe, however, did not cross-react with nucleic acid extracted from nine unrelated poultry viruses. A rapid procedure for isolation of IBDV genomic RNA from bursa and spleen tissue specimens was developed and used with the dot blot hybridization assay to detect IBDV strains in tissue samples from experimentally infected and commercially reared chickens.     

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.     

Effect of infectious bursal disease virus infection on the severity of Aspergillus flavus aspergillosis of chickens

Two groups of broiler chickens were infected with infectious bursal disease virus (IBDV) and Aspergillus flavus or A. flavus alone, respectively. Loss in weight was 2.5 times more severe in the IBDV + A flavus birds than in those infected with A. flavus alone. Mottling of liver with grey spots was observed only in the IBDV + A. flavus broilers. Histopathological sections of the lungs showed more necrosis, granulomas and fibrosis in the IBDV + A. flavus group, and the fungus was reisolated for a longer period from this group than from birds infected with A. flavus alone. These observations indicate that IBDV infection can increase the severity of A. flavus aspergillosis.     

Early occurrence of apoptosis in lymphoid tissues from chickens infected with strains of Newcastle disease virus of varying virulence

Newcastle disease virus (NDV), the causative agent of Newcastle disease, is a prevalent problem in the poultry industry and often the cause of severe economic loss. There are many strains of the virus and these have varying virulence. The most virulent strains cause systemic lesions of lymphoid tissues, with necrosis and severe lymphoid depletion. Less virulent strains do not cause as much necrosis, but may predispose to secondary infection with other pathogens. Apoptosis or programmed cell death, has been demonstrated to play a role in the pathogenesis of other paramyxovirus infections, notably those caused by measles and canine distemper viruses. To investigate the role of apoptosis in lymphoid organs during NDV infection, immunohistochemistry for determination of expression of caspase-3, a marker of imminent apoptosis, was performed on formalin-fixed paraffin wax-embedded tissues (spleen, thymus, caecal tonsils and bursa of Fabricius) from 4-week-old chickens infected with NDV strains of varying virulence 2 days previously. The amount of apoptosis was proportional to the severity of the clinical disease elicited by the strains.