Effect of bursal cell number on the pathogenesis of infectious bursal disease in chickens

The pathogenesis of infectious bursal disease (IBD) in chickens neonatally chemically bursectomized (CB) by cyclophosphamide and subsequently inoculated with various numbers of bursal cells was examined. CB chickens inoculated with at least 62.5 X 10(6) bursal cells were as susceptible to IBD clinical manifestations (as determined by gross and microscopic evaluation of bursal tissues, virus recovery from spleen, and antibody titer) as intact chickens following inoculation with virus at 5 weeks of age. In contrast, CB chickens inoculated with 2.5 X 10(6) or fewer bursal cells were refractory to the IBD clinical manifestations compared with intact chickens or CB chickens inoculated with 62.5 X 10(6) or more bursal cells. Results from this study suggest that the availability of a large number of bursal cells is an essential factor in the development of IBD.     

Prevention of avian lymphoid leukosis by induction of bursal atrophy with infectious bursal disease viruses.

Five groups of genetically susceptible chickens were inoculated at hatching with lymphoid leukosis virus; four of these were given infectious bursal viruses of varying virulence at 14 days of age and one group was not inoculated (control). All chickens in the control group developed evidence of lymphoid leukosis by 180 days. Two groups given relatively virulent bursal disease viruses, which destroyed bursal lymphoid cells, did not develop lymphoid leukosis. Treatment with avirulent vaccines had no visible effect on bursal morphology and did not significantly alter the incidence of lymphoid leukosis in two other groups, although the time of development was delayed. Results of our study show that viral-induced destruction of the bursa of Fabricius eliminates the development of lymphoid leukosis but that infection without bursal destruction has little effect on lymphoid leukosis.     

Morphologic changes in the bursa of fabricius of chickens after inoculation with infectious bursal disease virus.

Sequential morphologic changes in the bursa of Fabricius were studied after oral inoculation of 1-day-old chicks with infectious bursal disease virus (IBDV). The epithelial surface morphology was studied by scanning electron microscopy, whereas the IBDV replication was sequentially followed by immunofluorescence and transmission electron microscopy. The earliest detectable changes in the bursal epithelium were evident at postinoculation hour (PIH) 48. They were characterized by reduction in numbers and size of microvilli on the epithelial cells accompanied by gradual involution of the button-like bursal follicles. At PIH 96 some specimens showed localized surface erosions due to loss of epithelial cells. As the damage progressed, the infolding of the buttomlike follicles became more pronounced and the surface erosions became more extensive. Loss of surface epithelium exposed the underlying damaged bursal follicles which appeared to be bounded by columnar epithelium. Some follicles had lost almost all the lymphocytes and macrophages and appeared as empty craters. Intrafollicular replication of IBDV was detectable as early as PIH 24 by immunofluorescence technique. Viral replication primarily took place in the lymphoid follicles. Regeneration of the follicles was not seen up to postinoculation day 12, suggesting that the IBDV-induced bursal damage could be permanent.     

传染性法氏囊病病毒变异E株感染鸡细胞凋亡的研究

研究了传染性法氏囊病病毒（IBDV）变异E株人工感染28日龄SPF雏鸡后鸡法氏囊淋巴细胞的凋亡情况。电镜观察和DNA电泳分析结果表明,IBDV感染后12～48h,雏鸡法氏囊淋巴细胞出现典型细胞凋亡的形态学特征和生化特征;经流式细胞计检测和荧光染色观察,统计学分析表明,IBDV感染后24～48h,雏鸡法氏囊淋巴细胞凋亡数量显著增加（P<0.05或P<0.01）。试验结果揭示IBDV变异E株人工感染可以诱导雏鸡法氏囊淋巴细胞凋亡。     

Isolation, identification, and pathogenicity of two field strains of infectious bursal disease virus

Using a sentinel bird approach, two field isolates of infectious bursal disease virus (IBDV) were isolated from broiler farms in two major broiler-producing areas of the state of Georgia. These farms had a history of subclinical IBD associated with respiratory problems and poor performance. Isolates designated as U-28 and 3212 were isolated using specific-pathogen-free chicken embryos and chicken embryo bursal cells. These isolates were identified by means of agar gel precipitation and virus-neutralization tests, direct immunofluorescence, histopathology, and electron microscopy. Isolates U-28 and 3212 appear to differ in antigenicity and pathogenicity from previously known serotype I IBDV isolates. In evaluating the extent of bursal damage caused by these field isolates, an association was found between the bursa of Fabricius/body weight index, histopathology scoring of atrophy, and morphometric analysis of the total follicle area.     

Subclinical infectious bursal disease in commercial broiler flocks in Saskatchewan.

Five commercial broiler flocks, not vaccinated for infectious bursal disease virus, derived from infectious bursal disease virus-vaccinated breeder flocks were surveyed for evidence of bursal damage and infectious bursal disease virus infection. They were compared with two groups of birds raised in isolation. Serum samples from one day old chicks contained maternal anti-infectious bursal disease virus antibodies which declined to undetectable levels by four weeks of age. Serum antibody levels remained undetectable in both control groups and one commercial flock, whereas four of the five commercial flocks had actively produced anti-infectious bursal disease virus antibodies by slaughter age. The weight of bursae from infectious bursal disease virus-positive flocks declined as compared to controls after four weeks of age. The decline in weight correlated with the appearance of histopathological lesions. Infectious bursal disease virus antigen was demonstrated in selected infected bursae and infectious bursal disease bursae and infectious bursal disease virus was isolated from some of these damaged bursae. Clinical infectious bursal disease was not observed in any of the commercial flocks. The importance of subclinical bursal damage and immunosuppression is discussed.     

Differential modulation of cytokine, chemokine and Toll like receptor expression in chickens infected with classical and variant infectious bursal disease virus

ABSTRACT: Infectious bursal disease (IBD) is an important immunosuppressive disease of chickens. The causative agent, infectious bursal disease virus (IBDV), consists of two serotypes, 1 and 2. Serotype 1 consists of classic IBDV (cIBDV) and variant IBDV (vIBDV). Both of these strains vary in antigenicity and pathogenesis. The goal of this study was to compare the immunopathogenesis of cIBDV and vIBDV. Three-week-old specific pathogen free chickens were inoculated intraocularly with standard challenge strain (STC) (cIBDV) and a variant strain Indiana (IN) (vIBDV). The cIBDV produced more pronounced bursal damage, inflammatory response and infiltration of T cells as compared to vIBDV. There were significant differences in the expression of innate (IFN-α and IFN-β), proinflammatory cytokine and mediator (IL-6 and iNOS) in cIBDV- and vIBDV-infected bursas. The expression of chemokines genes, IL-8 and MIP-α was also higher in cIBDV-infected chickens during the early phase of infection. The expression of Toll like receptor 3 (TLR3) was downregulated at post inoculation days (PIDs) 3, 5, and 7 in the bursas of vIBDV-infected chickens whereas TLR3 was upregulated at PIDs 3 and 5 in cIBDV-infected bursas. In vIBDV-infected bursa, TLR7 expression was downregulated at PIDs 3 and 5 and upregulated at PID 7. However, TLR7 was upregulated at PIDs 3 and 7 in cIBDV-infected bursas. The expression of MyD88 was downregulated whereas TRIF gene expression was upregulated in cIBDV- and vIBDV-infected bursa. These findings demonstrate the critical differences in bursal lesions, infiltration of T cells, expression of cytokines, chemokines and TLRs in the bursa of cIBDV-and vIBDV-infected chickens.     

Development of Peyer's patch and cecal tonsil in gut-associated lymphoid tissues in the chicken embryo

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyer's patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckel's diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.     

Effect of administration of anti-bursal extract or perfusate serum on the antibody production in chickens

Antisera to bursal extracts or perfusates were prepared and the influence of such sera on antibody production in chickens was investigated by the injection of antisera during the embryonic stage. Antisera to cyclophosphamide treated bursal extracts or bursal perfusates were injected on the 15th day of embryogenesis. The level of antibodies produced by chickens treated by these antisera was equal to the controls but IgG antibodies were totally absent. These results suggested that the administration of these antisera inhibited the differentiation of IgM antibody producing cells.     

Cell surface immunoglobulin regulated checkpoints in chicken B cell development

The bursa of Fabricius is critical for the normal development of B lymphocytes in avian species. Productive colonization of bursal follicles by B cell precursors requires surface immunoglobulin expression. We have shown using retroviral gene transfer that expression of chimeric receptors containing the extracellular and transmembrane domains of murine CD8alpha and CD8beta fused to the cytoplasmic domains of chicken Igalpha and Igbeta can support productive bursal colonization in the chicken embryo in bursal cells lacking the expression of endogenous sIgM. We show here that chimeric receptor expression does not support continued bursal cell development after hatch. However intrabursal administration of anti-CD8 antibodies that ligate the CD8alpha:Igalpha chimeric receptor results in maintained numbers of bursal cells that express the chimeric receptor in the absence of endogenous sIgM. These results support a model in which sIgM receptor expression is required for productive bursal colonization in the chick embryo but sIgM receptor ligation is required to support later B cell development after hatch.     

采用继代鸡胚成纤维细胞生产鸡传染性法氏囊细胞苗的试验

采用原代鸡胚成纤维细胞和继代鸡胚成纤维细胞,生产了鸡传染性法氏囊细胞苗,效价(TCID50/mL)无明显差异.采用继代鸡胚成纤维细胞生产鸡传染性法氏囊细胞苗,减少种蛋消耗,降低劳动强度,增加产量,降低生产成本.     

Pathology and virus tissue distribution of Turkey origin reoviruses in experimentally infected Turkey poults

The pathogenesis of 4 isolates of turkey-origin reovirus (NC/SEP-R44/03, NC/98, TX/98, and NC/85) and 1 chicken-origin reovirus (1733) was examined by infecting specific pathogen free (SPF) poults. These turkey-origin reovirus (TRV) isolates were collected from turkey flocks experiencing poult enteritis and are genetically distinct from previously reported avian reoviruses. Microscopic examination of the tissues collected from the TRV-infected poults revealed different degrees of bursal atrophy characterized by lymphoid depletion and increased fibroplasia between the bursal follicles. To understand the relationship between virus spread and replication, and the induction of lesions, immunohistochemical staining (IHC) for viral antigen, in situ hybridization (ISH) for the detection of viral RNA, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for the detection of apoptosis in affected tissues was performed. Both IHC and ISH revealed viral antigen and RNA in the surface epithelial cells of the bursa, in macrophages in the interstitium of the bursa and, to lesser degree, in splenic red pulp macrophages and intestinal epithelial cells. Increased apoptosis of bursal lymphocytes and macrophages was observed at 2 and 5 days postinoculation. No lesions were found in tissues from poults inoculated with the virulent chicken-origin strain, however viral antigen was detected in the bursa and the intestine. Although all TRVs studied displayed similar tissue tropism, there were substantial differences in the severity of the lesions produced. Poults inoculated with NC/SEP-R44/03 or NC/98 had moderate to severe bursal atrophy, whereas poults inoculated with TX/98 or NC/85 presented a mild to moderate bursal lymphoid depletion. The lymphoid depletion observed in the bursa appears to be the effect of an indirectly induced apoptosis and would most likely result in immune dysfunction in poults infected with TRV.     

A comparison of immune responses to infection with virulent infectious bursal disease virus (IBDV) between specific-pathogen-free chickens infected at 12 and 28 days of age

Previous studies indicated that the pathogenesis of infectious bursal disease virus (IBDV) may vary depending on the age of the infected chicken. The reason for this difference is not clear, immunopathogenesis studies comparing different age groups are lacking. In the present study the IBDV-immunopathogenesis was compared in 12-day-old and 28-day-old specific-pathogen-free layer-type chickens. Birds were inoculated with the classic virulent strain IBDV-IM. Interestingly, the up-regulation of interferon (IFN)-gamma and interleukin-1beta expression varied between the two IBDV-infected age groups. The cytokine expression pattern was different between IBDV-infected age groups in magnitude and timing not only in bursal tissue and caecal tonsils but also in the spleen. No variations between the different age groups were observed in IBDV-induced bursa lesions, accumulation of intrabursal T cells and macrophages, and systemic stimulation of the release of nitric oxide inducing factors. Furthermore, virus-neutralizing antibody levels were comparable between age groups. Under these experimental conditions, age-related differences were found only for the cytokine patterns but interestingly this did not influence the outcome of the disease with IBDV-IM.     

海兰褐鸡法氏囊中Cu/Zn-SOD、Mn-SOD及NOS cDNA克隆及序列分析

通过对海兰褐鸡法氏囊中Cu/Zn-SOD、Mn-SOD及iNOS cDNA克隆及序列分析,了解海兰褐鸡法氏囊中主要氧化-抗氧化酶的分布及其基因组成,为研究传染性法氏囊病及与法氏囊相关禽病的氧化-抗氧化发病机制奠定基础.结果显示,Cu/Zn-SOD、Mn-SODcDNA序列分别出现2个碱基的突变,iNOS cDNA序列与原始序列同源性为100％.海兰褐鸡法氏囊中存在Cu/Zn-SOD、Mn-SOD及iNOS 3种与氧化-抗氧化相关酶,并且Cu/Zn-SOD、Mn-SOD cDNA序列具有新特征,iNOS在海兰褐鸡体内是稳定存在的;海兰褐鸡可能与康奈尔K系鸡具有相似的遗传背景.     

The Characterization of Infectious Bursal Disease Virus Strains/Isolates from Field Outbreaks in India

Pahar, B. and Rai, A., 1997. The characterization of infectious bursal disease virus strains/isolates from field outbreaks in India. Veterinary Research Communications, 21 (4), 289-301Three infectious bursal disease virus (IBDV) isolates were adapted to culture in chick embryo fibroblast cells in which they produced a cytopathic effect. The isolates were identified as IBDV by virus neutralization tests using a standard hyperimmune serum against infectious bursal disease, physicochemical properties and their pathogenicity in chick embryos and chicks. The IBDV S394 strain was antigenically different from IBDV S194/IBDV S494 as well as from the IBDV Intermediate Georgia strain, one of the vaccine strains in use in India.     

Organ culture of chicken bursa as a model to study the pathogenicity of infectious bursal disease virus isolates.

In vitro study with chicken bursal organ culture was attempted to assess the pathogenicity of locally isolated infectious bursal disease virus (IBDV) initially isolated from the bursa of naturally infected birds. In bursal organ culture, lymphoblastic transformation was noticed as early as 24 hr postinoculation and reached maximum at 72 hr postinoculation. The other microscopic changes were increased number of macrophages and formation of plasma cells. The IBDV antigen was detected 24 hr onward by coagglutination test with antibody coated Staphylococcus aureus strain Cowan I. On the basis of lesion score, the three isolates of IBDV (A, B, and C) were graded as virulent (B isolate) and moderately virulent (A and C isolates). A similar pattern of pathogenicity was also observed in the in vivo pathogenicity studies in chicken based on bursa: body weight ratio and histopathologic lesion score. The bursal organ culture thus provides a useful experimental model to differentiate the IBDV isolates on the basis of their virulence.     

Retrospective ultramicroscopic investigation on naturally cryptosporidial-infected commercial turkey poults

The morphometric characteristics and the ultramicroscopic findings of Cryptosporidium spp. at various stages of their life cycle in the intestinal and bursal epithelial cells of naturally infected 30-day-old commercial turkeys are reported. Small, sporulated oocysts, observed in the small intestinal content after flotation, were identified as Cryptosporidium meleagridis on the basis of morphometric characteristics (round in shape and 4.5-5.0 microm in size) and the small intestinal localization. Light section examinations revealed the presence of the protozoon in multiple organs, but its prevalence was highest in the intestinal and bursal epithelial cells. Ultramicroscopic studies on ileum and bursal samples showed the presence of all the life cycle stages in the microvillar brush epithelial cells in both the organs examined. On the basis of the comparison of the morphology and the sizes of the microorganisms parasitizing the ileum and the bursa, hypotheses are considered on the possible species involved.     

Chicken B lymphoma DT40 cells as a useful tool for in vitro analysis of pathogenic infectious bursal disease virus

Susceptibility of DT40 cells to pathogenic field strains of infectious bursal disease virus (IBDV) including very virulent and classical virulent strains were studied. After the first and second passage of the virus in DT40 cells, IBDV-specific antigen was readily detected in DT40 cells inoculated with the pathogenic field strain infected bursal homogenates. Nucleotide sequence analysis in the VP2 hypervariable domain, which is critical for the virulence of IBDV, revealed no common amino acid substitutions among the pathogenic IBDVs in accordance with the propagation in DT40 cells. These results indicate that DT40 cells are a useful tool for rapid isolation of pathogenic field strains and successive in vitro analysis of IBDV.     

IBDV-induced bursal T lymphocytes inhibit mitogenic response of normal splenocytes

We examined the suppressive activity of bursal T cells induced by infectious bursal disease virus (IBDV) in inbred (15x7) and outbred commercial specific-pathogen-free (SPF) chickens. The suppressive activity was measured by the ability of bursal and splenic T cells from IBDV-infected chickens to inhibit mitogenic responses of normal splenocytes. The bursacytes but not the splenocytes of IBDV-infected chickens inhibited the mitogenic responses of normal splenocytes. The mitogenic inhibition by the bursacytes of IBDV-infected chickens was dose-dependent. The suppression was observed both in inbred and non-inbred chickens, and thus, was non MHC-restricted. Cell-sorting experiments revealed that both CD4(+) and CD8(+) cells from the bursa of IBDV-infected chickens, as well as cell-culture supernatants conditioned by these cells, mediated suppression. Suppressor T (Ts) cells may therefore be involved in the immunosuppression induced by IBDV.