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.     

Functional restoration of the bursa of Fabricius following in ovo infectious bursal disease vaccination

The primary role of the avian bursa of Fabricius is to provide an essential microenvironment for B-lymphocytes to diversify their immunoglobulin genes by gene hyperconversion. Infectious bursal disease (IBD) vaccination using intermediate plus vaccine strains can temporarily deplete the bursal follicles and interrupt the normal B-cell development, which is generally followed by B-cell repopulation and histological regeneration. To find evidence that functional restoration of the bursa of Fabricius occurs in addition to the histological regeneration, we have analysed the chB1 gene expression, which indicates active bursal B-lymphocytes, and also the surface expression of a carbohydrate structure Lewis(x), a marker which identifies those bursal B-lymphocytes that are undergoing gene hyperconversion. In ovo vaccination with an immune complex vaccine (IBDV-BDA) caused transient bursal destruction in both the SPF and the maternally protected broiler groups with differences evident in the starting time, the severity and the duration of the effect. After the depletion phase, signs of histological regeneration appeared together with chB1- and Lewis(x) expression indicating that B-lymphocytes were functionally active and the bursa of Fabricius was serving again as an efficient primary lymphoid organ providing an appropriate microenvironment for B-cell development.     

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.     

Isolation and immunomodulatory activity of bursal peptide,a novel bursal peptide from the chicken bursa of Fabricius

The bursa of Fabricius (BF), which is unique to birds, serves as the central humoral immune organ and plays a significant role in B lymphocyte differentiation. In this study, a new bursal peptide (BP-IV) was isolated from BF, which promoted colony-forming unit pre-B formation and regulated B cell differentiation. BP-IV also exerted immunomodulatory effects on antigen-specific immune responses via both humoral and cellular immunity in chicken and mice that had been immunized with inactivated avian influenza virus (AIV; H9N2 subtype), including enhancing AIV-specific antibody and cytokine production. The results of this study provided novel insights into the use of a potential candidate reagent for B cell development and future immuno-pharmacological use.     

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.     

Protection against infectious bursal disease virulent challenge conferred by a recombinant avian adeno-associated virus vaccine

The development and use of recombinant vaccine vectors for the expression of poultry pathogens proteins is an active research field. The adeno-associated virus (AAV) is a replication-defective virus member of the family Parvoviridae that has been successfully used for gene delivery in humans and other species. In this experiment, an avian adeno-associated virus (AAAV) expressing the infectious bursal disease virus (IBDV) VP2 protein (rAAAV-VP2) was evaluated for protection against IBDV-virulent challenge. Specific pathogen free (SPF) birds were inoculated with rAAAV-VP2 or with a commercial intermediate IBDV vaccine and then challenged with the Edgar strain. IBDV-specific antibody levels were observed in all vaccinated groups; titers were higher for the commercial vaccine group. The live, commercial vaccine induced adequate protection against morbidity and mortality; nevertheless, initial lymphoid depletion and follicular atrophy related to active viral replication was observed as early as day 14 and persisted up to day 28, when birds were challenged. No bursal tissue damage due to rAAAV-VP2 vaccination was observed. Eight-out-of-ten rAAAV-VP2-vaccinated birds survived the challenge and showed no clinical signs. The bursa:body weight ratio and bursa lesion scores in the rAAAV-VP2 group indicated protection against challenge. Therefore, transgenic expression of the VP2 protein after rAAAV-VP2 vaccination induced protective immunity against IBDV challenge in 80% of the birds, without compromising the bursa of Fabricius. The use of rAAAV virions for gene delivery represents a novel approach to poultry vaccination.     

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.     

Effects of estrogen on estrogen receptor expression in the bursal cells of chick embryos and steroidogenic enzymes gene expression in the bursa: Relevance of estrogen receptor and estrogen synthesis in the bursa of chick embryos

Estrogen has been reported to act on B cell genesis in the bursa of Fabricius of chick embryos. In this study, we attempted to demonstrate the hypothesis that B cell genesis is controlled by estrogen receptor (ER) in the bursal cells and steroidogenic enzymes synthesized in the bursa. We previously reported the presence of estrogen receptor α (ERα) in the bursa during the late stage of embryogenesis and an increase in the expression of ERα messenger RNA (mRNA) between the 13th day and 16th day. The number of ER-positive cells was maximal on the 16th day. In the present study, ER-positive cells in the bursa during the late stage of embryogenesis increased 4 h after β-estradiol treatment on the 14th to 18th day. The concentration of β-estradiol in the embryonic bursa increased. These results suggest that this stage of embryogenesis is critical in B cell development in the bursa in connection with the effect of estrogen treatment. Our findings also showed that the mRNA expression of five steroidogenic enzymes occurred in the bursa of chick embryos. These results suggest that estrogen is synthesized in the embryonic bursa and estrogen acts on the bursal cells in a paracrine fashion.     

In vitro infection of fractionated chicken lymphocytes by infectious bursal disease virus

Lymphocytes from bursa of Fabricius and thymus of chickens were purified and separated into the three cell subsets--T, B, and null cells--by the techniques of nylon fiber columns and cytotoxicity tests. The in vitro susceptibility of the fractionated lymphocytes to a virulent strain of infectious bursal disease virus (IBDV) was studied by using immunofluorescence as the infection criterion. B cells were highly susceptible. By contrast, T cells and null cells were insusceptible to infection by IBDV. The relationship between the target cells for virus infection and those B cells that possessed surface immunoglobulin (SIg) was tested. B cells were further divided into SIg(M)- and SIg(G)-bearing cells by immunoadsorbent columns employing anti-immunoglobulin M(IgM) (mu-specific) or anti-IgG (gamma-specific) sera coated with Sephadex. The SIg(M)-bearing cells were highly susceptible. These results suggest strongly that SIg(M)-bearing B cells were the target cells for infection by IBDV.     

B cell and macrophage response in chicks after oral administration of Salmonella typhimurium strains

Despite the fact that, in a number of countries, vaccination programmes are extensively used to control Salmonella infection in poultry, information on the immune mechanisms, especially the cellular response, is still needed. The aim of the study was to characterise the B cell and macrophage response in caecum (IgA+, IgM+, IgG+ cells, macrophages), bursa of Fabricius (IgM+ cells, macrophages), and spleen (IgM+ cells) of chicks after oral administration of a non-attenuated Salmonella (S.) typhimurium wild-type strain (infection) or an attenuated commercial live S. typhimurium vaccine strain (immunisation) to day-old chicks as compared to non-treated control birds using immunohistochemistry and image analysis. In caecum, higher counts of IgM-secreting cells were detected in infected animals compared with the controls from day 5 until day 12 of age. In contrast, in treated groups, IgA-secreting cells were found in higher numbers only between day 8 and 12 of age. Infected birds showed a higher number of IgA+ cells in spleen and bursa of Fabricius compared to the controls. In the bursa of Fabricius of immunised and infected birds, a depletion of strongly stained IgM+ cells and macrophages was established between day 5 and 9 indicating a possibly special and independent role of this organ during the immunological reaction against Salmonella organisms. The results suggest that IgM- and IgA-secreting cells are of importance in the caecal immune response of chickens against Salmonella strains. Immunised chickens always showed a weaker immune reaction compared to infected animals. Present findings regarding the B cell reaction within avian caeca prove a participation of both humoral and cellular immunity in defence against Salmonella strains. Immunohistochemical examination of the cellular response (B cells and macrophages) in relevant organs of chickens may be an important tool to evaluate the immunogenic characteristics of potential Salmonella live vaccine candidates.     

A B-cell targeting virus disrupts potentially protective genomic methylation patterns in lymphoid tissue by increasing global 5-hydroxymethylcytosine levels

The mechanisms by which viruses modulate the immune system include changes in host genomic methylation. 5-hydroxymethylcytosine (5hmC) is the catalytic product of the Tet (Ten-11 translocation) family of enzymes and may serve as an intermediate of DNA demethylation. Recent reports suggest that 5hmC may confer consequences on cellular events including the pathogenesis of disease; in order to explore this possibility further we investigated both 5-methylcytosine (5mC) and 5hmC levels in healthy and diseased chicken bursas of Fabricius. We discovered that embryonic B-cells have high 5mC content while 5hmC decreases during bursa development. We propose that a high 5mC level protects from the mutagenic activity of the B-cell antibody diversifying enzyme activation induced deaminase (AID). In support of this view, AID mRNA increases significantly within the developing bursa from embryonic to post hatch stages while mRNAs that encode Tet family members 1 and 2 reduce over the same period. Moreover, our data revealed that infectious bursal disease virus (IBDV) disrupts this genomic methylation pattern causing a global increase in 5hmC levels in a mechanism that may involve increased Tet 1 and 2 mRNAs. To our knowledge this is the first time that a viral infection has been observed to cause global increases in genomic 5hmC within infected host tissues, underlining a mechanism that may involve the induction of B-cell genomic instability and cell death to facilitate viral egress.     

Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands

Cells of the adaptive immune system express Toll-like receptors (TLRs) and are able to respond to TLR ligands. With this in mind, the goal of the current study was to determine the expression of antiviral response genes in the cells of the chicken bursa of Fabricius (BF) to stimulation with TLR ligands. We investigated initially the response of bursal B cells to CpG-ODN, lipopolysaccharide (LPS) and poly(I:C) treatment. The expression level of type I interferons (IFNs) and interferon regulatory factor 7 (IRF7) did not differ between CpG-ODN and LPS treated groups compared to the non-stimulated cells. Poly(I:C) was the only TLR ligand, which has induced significant expression of antiviral innate immune response genes from bursal cells. Further in vitro and in vivo studies need to examine the efficacy of these antiviral responses against avian viruses.     

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.     

An area of T cell localization in the cloacal bursa of White Leghorn chickens

Within the cloacal bursa (bursa of Fabricius), there is a diffusely infiltrated area (DIA) of lymphoid cells just dorsal to the bursal duct opening. Because this region displayed certain histologic characteristics of thymus-dependent areas found in mammals, such as high endothelial venules, heavy reticular fiber components, and epithelium infiltrated with lymphocytes, experiments were conducted to determine whether thymus-derived cells (T cells) were present. Two independent methods substantiated the presence of T cells in the DIA. After the acid alpha-naphthyl acetate staining technique, the characteristic T cell focal esterase activity could be demonstrated in significant numbers of cells found in the DIA. Also autologous radioactively labeled thymocytes could be identified in the DIA by autoradiography a few hours after IV injection. This establishment of a definite T cell area within the bursa gives additional credence to the contention that the bursa functions as a secondary lymphatic organ.     

Effect of ascorbic acid supplementation on the immune response of chickens vaccinated and challenged with infectious bursal disease virus

One-day-old chickens were divided into two groups and reared under similar conditions. One group was fed a diet supplemented with 1000ppm ascorbic acid and the other group was fed an identical diet, but not supplemented with ascorbic acid. Both groups were vaccinated against infectious bursal disease (IBD) at 7 days of age and challenged orally with 4x10(5) of 50% embryo-lethal-dose IBDV 14 days later. The number of anti-IBDV antibody secreting cells, production of interleukin-2 (IL-2) by splenocytes, number of CD4(+), CD8(+) and IgM(+) cells in spleen and IgM(+) cells in bursa of Fabricius were compared between the two groups at 7 days (prior to vaccination), 21 days (14 days post-vaccination and prior to challenge) and 31 days (10 days post-challenge) of age. The number of CD8(+) in spleen at 7 days of age and IgM(+) cells in bursa at 7, 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Production of IL-2 by splenocytes was higher as indicated by higher stimulation indices in ascorbic acid supplemented group. The number of anti-IBDV IgG antibody secreting cells in spleen at 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Dietary supplementation of ascorbic acid may ameliorate the immunosuppression caused by IBDV vaccination and improve humoral and cellular immune responses.     

禽网状内皮组织增生病病毒感染对SPF雏鸡法氏囊细胞Bcl-2和C-myc基因表达及凋亡的影响

【目的】 探究禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)感染禽类后导致免疫器官发生细胞凋亡的机理。【方法】 以1日龄SPF雏鸡为试验对象,将100只SPF雏鸡随机均分为REV感染组和未感染病毒的对照组,REV感染组雏鸡经腹腔感染500 μL REV稀释液,对照组雏鸡经相同途径注射等量灭菌生理盐水,于病毒感染后第1、7、14、21、28和42天,2组雏鸡随机各抽取5只,心脏采血处死雏鸡后快速摘取法氏囊。分别应用HE染色和病理切片成像系统测定分析法氏囊细胞核浆比,TUNEL细胞凋亡原位检测试剂盒测定凋亡细胞数,免疫组化法测定Bcl-2和C-myc阳性细胞数量,实时荧光定量PCR和ELISA法分别检测法氏囊Bcl-2和C-myc 基因mRNA表达和蛋白含量。【结果】 ①REV感染1日龄SPF雏鸡后21~42 d,其法氏囊淋巴细胞凋亡百分比显著或极显著高于对照组雏鸡(P<0.05;P<0.01);②SPF雏鸡感染REV后21和28 d,其法氏囊细胞核浆比显著低于对照组雏鸡(P<0.05);③REV感染SPF雏鸡法氏囊中Bcl-2和C-myc阳性细胞数在病毒感染后21和28 d显著高于对照组雏鸡(P<0.05);④REV感染SPF雏鸡后21 d,其法氏囊Bcl-2和C-myc 基因mRNA表达极显著高于对照组雏鸡(P<0.01)。⑤SPF雏鸡感染REV后,其法氏囊中Bcl-2蛋白含量较对照组雏鸡有不同程度的增加,其中21和28 d分别差异极显著(P<0.01)和显著(P<0.05),病毒感染组雏鸡的C-myc蛋白含量也始终高于对照组雏鸡,且21和28 d极显著增高(P<0.01)。【结论】 REV感染所致SPF雏鸡法氏囊细胞Bcl-2和C-myc的mRNA表达以及蛋白含量异常均与病毒感染导致的法氏囊细胞凋亡密切相关,而法氏囊细胞凋亡数量增加与REV感染引发的机体免疫机能抑制密切相关。     

鸡贫血病—法氏囊病疫苗免疫母鸡后子代雏鸡的免疫学变化

本项目应用现代免疫学新技术对鸡传染性贫血病（CIA）-传染性法氏囊病（IBD）疫苗联合免疫母鸡后,其子代雏鸡外周血液T、B细胞数量和IgG、IgM、IgA含量法及法氏囊、胸腺、脾脏、盲肠扁桃体、哈德尔腺的T细胞和IgG、IgM、IgA抗体生成细胞数量以及泪液、气管液、胆汁、肠液的IgA、IgM、IgG含量的变化进行了动态研究。结果发现,CIA-IBD疫苗联合免疫母鸡后,其子代雏鸡外周血液、免疫器官组织和局部体液的上述各项指标均不同程度地高于未免疫的相应对照雏鸡。表明CIA-IBD疫苗免疫母鸡后,其子代雏鸡的体液免疫和细胞免疫功能明显增强,而CIAV-IBDV强毒攻击后,未免疫的子代雏鸡,其外周血液,免疫器官组织和局部体液的各项免疫学指标均明显低于疫苗免疫攻毒的子代雏鸡,这与未免疫雏鸡缺乏特异性抗体,强毒攻击后,雏鸡免疫器官组织广泛损害,淋巴细胞变性坏死等有关。     

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.     

传染性法氏囊病病毒免疫学研究进展

传染性法氏囊病病毒(IBDV)母源抗体对雏鸡的保护期大约为3周,在自然条件下,大多数鸡在2周龄～15周龄都能够感染IBDV.IBDV通过口腔或者呼吸道方式进入机体,在肠道相关组织的淋巴细胞和巨噬细胞内复制,然后通过血液循环进入法氏囊,盲肠扁桃体的细胞因子表达在强毒攻入SPF鸡体内后发生变化即是很好的例证.体内外的研究表...