Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Abstract. Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stage as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Hematopoietic differentiation cell surface antigen switching in the bone marrow of different aged chickens

Immune cytolysis and immunofluorescence were used to examine chicken fetal antigen CFA) and chicken adult antigen (CAA) expression on the differentiation/maturation series of definitive erythroid cells obtained from the bone marrow of different aged chickens. We found that erythroid cells undergo changes in CFA/CAA antigenic expression dependent on their differentiation/maturation stages as well as the developmental age of the chicken. All differentiation/maturation stages of erythroid cells in the bone marrow of 12 and 18-day-old embryos express CFA only. Erythroblasts obtained from 7-day post-hatched chickens express either CFA or CAA. All three CFA/CAA phenotypes (i.e., CFA, CAA, and CFA + CAA) are observed in subsequent maturation stages, but only the CFA + CAA phenotype is observed in mature erythroid cells in the bone marrow of 7-day post-hatched chickens. Erythroblasts from 62 day post-hatched chickens exhibit all three CFA/CAA phenotypes. Cells in the subsequent maturation stages express various CFA, CAA, or CFA + CAA phenotypes resulting in a majority of the mature erythrocytes expressing both CFA and CAA, and a small population of mature erythrocytes expressing CAA only. Erythroblasts from adult chickens express both CFA and CAA; however, CFA is lost during erythroid maturation resulting in mature erythrocytes which express CAA only. These studies indicate that both the erythroid differentiation/ maturation stage and the developmental age of the chicken influence CFA and CAA antigenic expression on erythroid cells undergoing cellular differentiation/maturation in the bone marrow.     

Chicken developmental antigens: analysis of erythroid populations with monoclonal antibodies

Fusions were performed between the mouse PAI myeloma cell line and spleen cells from Balb/c mice immunized with intact erythrocytes from 1-day Cornell K-strain White Leghorn chickens. Following single cell cloning, four hybridoma clones were found to secrete erythroid specific monoclonal antibodies. Based on its pattern of reactivity, the antibody (IgG2a, kappa) secreted by clone 10C6 detects a specific avian oncodevelopmental antigen associated with the hematopoietic system: chicken fetal antigen (CFA). Two other clones, designated as 3F12 and 4C2, produced antibodies (IgM, kappa) that recognize another avian developmental antigen: chicken adult antigen (CAA). A fourth clone, 9F9, produced an antibody (IgM, kappa) that reacts with all peripheral erythrocytes from both Japanese quail and chicken regardless of age. Clone 10C6 antibody apparently detects an erythrocyte specific (ES) determinant of CFA associated with determinant #8 while antibodies of clones 3F12 and 4C2 recognize a chicken specific determinant of CAA. Analysis by complement mediated microcytotoxicity indicated that the epitopes detected by 10C6 vs 3F12 and 4C2 antibodies were expressed on erythrocytes in a reciprocal fashion during development. Furthermore, strain variations in the incidence of erythrocytes carrying these epitopes were observed. The usefulness of these monoclonal antibodies for the study of erythroid populations is discussed.     

Avian developmental antigens: Expression on erythrocytes of chickens,Japanese quail,and quail-chicken hybrids

Monoclonal and polyclonal antibodies were used to examine the expression of three erythroid developmental antigen systems in the chicken, Japanese quail, and quail-chicken hybrid. Chicken fetal antigen (CFA), quail fetal antigen (QFA), and chicken adult antigen (CAA) each represent a series of cell-surface glycorproteins associated with the development of avian hematopoietic cells. Monoclonal anti-CFA antibodies from clones 190-4 and 288-1.1.1.2 supernatants were shown to react against epitopes associated with CFA determinants 8 and 2, respectively. Using complement-mediated microcytotoxicity, these reagents permitted the identification of different erythroid subpopulations in the neonatal chicken and hybrid; therefore, heterogeneity in cell surface CFA determinants among mature peripheral erythrocytes should serve as a useful tool for analyzing erythroid development. In the case of CAA, erythrocytes from adult hybrids were found to express the same complement of CAA determinants identified in the chicken, and CAA appeared much earlier in the hybrid than in either of the parental species. Similarly, two species-restricted fetal antigens associated with similar glycoproteins, CFA8 and QFA, had similar developmental profiles in their respective species, the chicken and quail. In contrast, these antigens were dominantly expressed but exhibited different developmental profiles on erythrocytes from the hybrids. While quail-chicken hybrids exhibited apparent genomic interactions in the expression of these developmental antigens, no evidence for the existence of hybrid-specific fetal antigens was obtained.     

Chemical and immunological characterization of developmentally expressed chicken erythroid surface membrane antigens

The expression of two hematopoietic-lymphoid membrane antigens, referred to as chicken fetal antigen (CFA) and chicken adult antigen (CAA) were investigated on primitive and definitive peripheral red blood cells (RBC) from different-aged chickens using chemical and immunological techniques. Differential adsorptions of antisera specific for adult RBC membrane antigens permitted the serological dissection of CAA into eight antigenic determinants. CFA and CAA were assayed by hemagglutination, hemolysis, and immune precipitation of radioiodinated surface antigens of RBC from different-aged chickens. Primitive RBC express CFA, while definitive RBC express three distinct phenotypes: CFA, both CFA and CAA, or CAA, depending on the developmental age of the chicken from which the RBC were obtained. When solubilized membrane extracts of radioiodinated peripheral RBC from chickens at 5 and 18 days embryonic development (E5 and E18, respectively), 13 days posthatch development (H13), and adult chickens were immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the major antigen detected by anti-CFA sera was associated with proteins having apparent molecular weights (M_r) of 50,000 daltons (50 kd). The antigens detected by anti-CAA sera were associated with proteins having apparent M_r of 102, 81, 48, and 43 kd.     

Genetic variation in the recruitment and activation of chicken peritoneal macrophages

Genetic variation in the ability to recruit and activate peritoneal macrophages was examined in seven partially developed 15I5-B congenic White Leghorn chicken lines. While the ability to generate peritoneal exudate cells (PECs) was similar in all lines, major differences were observed in the numbers, composition, and functional activity of harvestable peritoneal adherent cell populations. In response to a general stimulant, Sephadex, lines .7-2 and .6-2 produced the greatest numbers of adherent peritoneal cells while lines .C-12 and .15I-5 were among the poorest responders. Macrophage percentage of adherent PECs varied between lines. 15I5 chickens produced a consistently high percentage of adherent macrophages while .6-2 birds exhibited the lowest macrophage percentage at all ages examined. Phagocytosis was used as one measure of the level of macrophage activation and similar results were obtained using both opsonized and unopsonized sheep erythrocytes; adherent peritoneal cells from lines .6-2, .7-2, and .P-13 exhibited the highest activity and .C-12, .15I-5, and background 15I5(B15) lines produced cells with the lowest phagocytic activity. In a second functional assay, the killing of Salmonella typhimurium, macrophage-rich cells from line .P-13 exhibited the lowest activity which was significantly lower than that obtained with cells from lines .6-2 and .15I-5. Antigen-specific stimulation of peritoneal adherent cells by ferritin also showed that .C-12 was a low responder in contrast with other lines. The results indicate that these genetic lines differ in peritoneal macrophage function and suggest that the chicken major histocompatibility complex may influence certain properties of chicken macrophage function.     

Evidence for multiple cell surface chicken fetal-leukemic antigens (CFA) in the developing chick and other avian species.

Chicken fetal antigen (CFA), a membrane antigen present on fetal chicken red blood cells is lost with chicken development, and reappears on the red blood cells of leukemic chickens. Seven avian species were found to possess CFA. A species hierarchy comparing the quantitative expression of CFA has been established. The levels of CFA expression with development are compared in the chicken and Japanese quail. Specific adsorptions of R-anti-CFA with avian red blood cells revealed the existence of multiple CFAs. Four groups of antigenic determinants (CFA a,b,c,d) have been characterized and defined by their expression among avian species. Multiple CFA determinants are discussed with regard to possible membrane alterations and gene function.     

Chicken fetal antigen: association with lymphoid development and differentiation

Rabbit antisera capable of detecting chicken fetal antigen (CFA) was prepared against 1-day chick red blood cells (RBCs) and made specific by exhaustive adsorption with adult chicken peripheral RBCs (PRBCs) from the same strain. Microcytotoxicity was used to study the incidence of CFA on lymphocytes obtained from several organs at different developmental stages in the chicken. Lymphocyte-associated CFA (LA-CFA) determinants and erythrocyte-specific CFA (ES-CFA) determinants were distinguished through the use of adsorption analysis. The high incidence of CFA-positive lymphocytes found in the fetal bursa and thymus was not equaled in the peripheral organs of the spleen, cecal tonsils, and gland of Harder. CFA expression on adult lymphocytes was restricted to the thymus and peripheral blood. It is suggested that these cells may represent a subpopulation of T lymphocytes. Adult spleen, cecal tonsils, and gland of Harder were virtually devoid of CFA-bearing lymphocytes. At fetal developmental stages when greater than 94% of the bursal B cells were CFA-positive, few, if any, of the highly differentiated Harderian B cells possessed CFA. It is suggested that LA-CFA expression is dependent upon B cell differentiation and/or the bursa (central) vs gland of Harder (peripheral) microenvironment. The pattern of CFA expression on bursacytes is discussed in light of the properties of age resistance and bursal-dependent target cells associated with virally induced lymphoid leukosis.     

Isolation of chicken anemia agent with MDCC-MSB1 cells from chickens in the field

An attempt was made to isolate chicken anemia agent (CAA) from chickens suffering from anemia in the field by using MDCC - MSB1 , which was an established cell line derived from Marek's disease lymphoma. When 99 chickens of 15 flocks were examined, CAA was isolated from 58 chickens of 12 flocks. The rate of CAA isolation with MDCC - MSB1 cells was almost the same as that determined by an in vivo method by chick inoculation. It was shown that CAA was more closely concerned with anemic diseases of chickens in the field than fowl adenoviruses.     

Murine embryonic antigen (SSEA-1) is expressed on human cells and structurally related human blood group antigen I is expressed on mouse embryos

The stage-specific embryonic antigen (SSEA-1), present on embryonal carcinoma cells and on murine preimplantation embryos, is defined by a monoclonal antibody. The antigenic determinant of SSEA-1 is a carbohydrate structurally related to the human blood group antigen I. Since it has been suggested that the I antigen might represent a precursor or SSEA-1, we used antibodies to SSEA-1 and to I to analyze their expression on mouse preimplantation embryos. Both are expressed on mouse embryos; moreover, I is expressed on earlier embryos than SSEA-1. The I antigen is defined by its expression on human erythrocytes; accordingly, we examined expression of I and SSEA-1 on human peripheral blood elements. We find SSEA-1 to be expressed exclusively on human granulocytes while I is found only on erythrocytes. These results suggest that these closely related antigens can be independently expressed. Analysis of the expression of I and SSEA-1 was then extended to a series of mouse and human cell lines; some express both, some express only one, and some express neither of these antigens. The activation of specific glycosyltransferases and/or glycosidases during development and differentiation appears to be the biochemical mechanism regulating expression of these antigens.     

双歧杆菌表达ETEC CFA／I的免疫电镜定位分析

目的研究双歧杆菌表达的CFA／I在细胞中的分布。方法将培养8～10h的重组双歧杆菌固定后制作电镜切片，镜检合格后用镍网捞取3-5个切片与金标抗体作用，镜检CFA／I在双歧杆菌中的表达分布。结果自制的胶体金颗粒均匀，与抗体结合后能够示踪CFA／I在双歧杆菌中的表达分布。结论双歧杆菌表达的CFA／I主要集中在靠近细胞膜的周质腔一侧，利于向外分泌和释放。     

Age-dependent changes in cation transport in the chicken erythrocyte

Previous work has shown that the transport phenotype of chicken erythrocytes changes with the age of the chicken. Here, we report changes in the transport of choline and K+ in erythrocytes from chickens at different developmental ages. The transport of choline in chicken erythrocytes was predominantly via saturable transport systems, was highest in erythrocytes from 1-day-old chickens and declined with chicken age when tested at 2 weeks of age and in mature chickens. Both Km and Vmax values for choline transport in chicken erythrocytes declined with chicken age. Similarly, the total unidirectional influx of K+ was highest in erythrocytes from 1-day-old chickens and declined with chicken age, as did ouabain-sensitive K+ influxes, which can be attributed to the Na+/K+ pump. In isotonic conditions, bumetanide-sensitive K+ influxes, which can be attributed to the Na+-K+-2Cl- cotransporter, were only measurable in erythrocytes from 1-day-old chickens. However, when stimulated by hypertonic conditions, bumetanide-sensitive K+ influxes were essentially identical in erythrocytes from 1-day- and 2-week-old chickens but decreased in erythrocytes from mature chickens. We conclude that both choline and K+ influxes decrease significantly in erythrocytes from chickens with increasing age. The changes are substantial but complex and may involve both regulation of existing transporters, and substitution or deletion of specific transporter isoforms.     

表达H5亚型禽流感病毒HA基因和鸡IL-18基因重组禽痘病毒的构建

[目的]获得共表达H5亚型AIV HA基因和鸡IL-18基因的重组禽痘病毒.[方法]将含痘病毒启动子LP2EP2的HA基因和鸡IL-18基因插入到禽痘病毒转移载体pSY681中,获得重组转移载体pSYHA/IL-18.用脂质体将其转染已感染亲本禽痘病毒S-FPV-017株的鸡胚成纤维细胞,使其在鸡胚成纤维细胞内与禽痘病毒基因组发生同源重组,产生表达HA和IL-18的重组禽痘病毒(rFPV-HA-IL-18).在含有X-gal的营养琼脂培养基上进行蓝斑筛选后,对重组禽痘病毒又进行了多次蚀斑克隆.[结果]以重组禽痘病毒DNA为模板,利用HA基因和鸡IL-18基因引物进行PCR,分别扩增出1条约1.7 kb带和1条0.6 kb左右的带.以间接免疫荧光试验、T细胞转化试验和SPF雏鸡免疫接种证实重组禽痘病毒能表达HA和鸡IL-18,并初步证明鸡IL-18增强HA免疫作用.[结论]重组禽痘病毒能表达具有生物学活性的HA和鸡IL-18.     

Genetic regulation of CFA expression in interspecific avian hybrids and somatic cell hybrids

Chicken fetal-leukemic antigen (CFA) is an oncodevelopmental antigen present on embryonic and neonatal chicken peripheral red blood cells (RBCs) but is not restricted to fetal stages of development in other avian species. Crosses between white Leghorn chickens and Japanese quail resulted in adult hybrids whose peripheral RBCs were positive for CFA. Of the four CFA determinants normally found in adult quail RBCs, only two were present on quail-chicken hybrid RBCs. Adult quail--chicken hybrid RBCs also possessed on CFA determinant associated with early development in both quail and chicken and one chicken-specific CFA determinant. Evidence is presented for the possible association of CFA-positive adult peripheral RBCs and the level of circulating reticulocytes. Crosses between pheasant and turkey (both with CFA-positive adult RBCs) resulted in hybrid adult RBCs expressing only a portion of the parental CFA determinants. Through the formation of somatic cell hybrids between adult chicken and embryonic Japanese quail RBCs, it was possible to induce the appearance of CFA determinants normally restricted to embryonic chicken RBCs. Approximately 50% of the hybrid cells showed reexpression of CFA, and this induction was both time and temperature dependent. Hybridization between RBCs of adult chicken and those of either adult Japanese quail or adult turkey failed to elicit the reexpression of chicken-specific CFA.     

Characterization of colonization factor antigen CFA/I from an enterotoxigenic Escherichia coli strain (0128:H12)

CFA/I antigen was isolated and purified from E. coli, mutant 279 B-1-14, serotype 0128:H12, and had the following biochemical and biological features: a) amino-acid content was similar to that of purified antigen prepared from strain H10407; b) latex particles sensitization with purified CFA/I antigen produced bovine and human erythrocytes group A/II hemagglutination in carbohydrates presence; c) purified anti-CFA/I specific antibodies agglutinated CFA/I-positive enterotoxigenic E. coli strains; d) 3H-leucine-labelled CFA/I antigen adhered to rabbits intestinal mucosa at significant values; e) intestinal mucosa pretreating with purified CFA/I antigen, followed by 3H-leucine labelled enterotoxigenic bacteria infection, had a least 3 local effects: 1) intestinal mucosa protection against parental enterotoxigenic bacteria; 2) inhibition of CFA/I-positive bacteria adherence to intestinal mucosa; 3) release of approximately 96% intraluminally inoculated bacteria.     

Co-administration of avian influenza virus H5 plasmid DNA with chicken IL-15 and IL-18 enhanced chickens immune responses

ABSTRACT: BACKGROUND: DNA vaccines offer several advantages over conventional vaccines in the development of effective vaccines against avian influenza virus (AIV). However, one of the limitations of the DNA vaccine in poultry is that it induces poor immune responses. In this study, chicken interleukin (IL) -15 and IL-18 were used as genetic adjuvants to improve the immune responses induced from the H5 DNA vaccination in chickens. The immunogenicity of the recombinant plasmid DNA was analysed based on the antibody production, T cell responses and cytokine production, following inoculation in 1-day-old (Trial 1) and 14-day-old (Trial 2) specific-pathogen-free chickens. Hence, the purpose of the present study was to explore the role of chicken IL-15 and IL-18 as adjuvants following the vaccination of chickens with the H5 DNA vaccine. RESULTS: The overall HI antibody titer in chickens immunized with pDis/H5 + pDis/IL-15 was higher compared to chickens immunized with pDis/H5 (p < 0.05). The findings revealed that the inoculation of the 14-day-old chickens exhibited a shorter time to achieve the highest HI titer in comparison to the inoculation of the 1-day-old chickens. The cellular immunity was assessed by the flow cytometry analysis to enumerate CD4+ and CD8+ T cells in the peripheral blood. The chickens inoculated with pDis/H5 + pDis/IL-15 demonstrated the highest increase in CD4+ T cells population relative to the control chickens. However, this study revealed that pDis/H5 + pDis/IL-15 was not significant (P > 0.05) in inducing CD8+ T cells. Meanwhile, with the exception of Trial 1, the flow cytometry results for Trial 2 demonstrated that the pDis/H5 + pDis/IL-18 inoculated group was able to trigger a higher increase in CD4+ T cells than the pDis/H5 group (P < 0.05). On the other hand, the pDis/H5 + pDis/IL-18 group was not significant (P > 0.05) in modulating CD8+ T cells population in both trials. The pDis/H5 + pDis/IL-15 inoculated group showed the highest IL-15 gene expression in both trials compared to other inoculated groups (P < 0.05). Similar results were obtained for the IL-18 expression where the pDis/H5 + pDis/IL-18 groups in both trials (Table 8) were significantly higher compared to the control group (P < 0.05). However, the expressions of other cytokines remained low or undetected by GeXP assay. CONCLUSIONS: This study shows the diverse immunogenicity of pDis/H5 co-administered with chicken IL-15 and IL-18,with pDis/H5 + pDis/IL-15 being a better vaccine candidate compared to other groups.     

Immunochemical analysis of histone H1 and H5 from pigeon erythroid cells]

An antiserum with the antibody titer of 1 : 4096 was obtained by immunization of rabbits with the tRNA-histone H5 complex from pigeon erythrocytes. The specificity of the antiserum was studied quantitatively from the reaction of the complement binding to a homologous antigen (histone H5) and its modifications (I, II, III), differing in the degree of phosphorylation. It was shown that phosphorylation of histone H5 increases the ability of the antigen to bind to antibodies, which is especially well-pronounced at the antiserum dilutions as high as 20480. The comparison of the antigenic properties of histones H5 from pigeon and chicken erythrocytes revealed beside structural differences of the proteins the presence of common antigenic determinants. A similar observation was made when histones H5 and H1 from pigeon erythrocytes were compared. Histone H1 from chicken erythrocytes and histone H1 from calf thymus did not produce criss-cross reactions with antiserum H5.     

Development and characterization of a recombinant chicken single-chain Fv antibody detecting <Emphasis Type="Italic">Eimeria acervulina</Emphasis> sporozoite antigen

Chicken monoclonal antibody (mAb), 8C3, which is reactive with a sporozoite antigen of Eimeria acervulina, is a potential therapeutic agent against avian coccidiosis caused by Eimeria spp. However, production of large amounts of 8C3 mAb in cell culture system is labor intensive and not cost-effective. Accordingly, recombinant single chain variable fragment (ScFv) antibody was constructed by amplification of the V_H and V_L genes from chicken hybridoma, 8C3 and when expressed in E. coli gave 5 mg l^–1. The expressed protein showed antigen binding activity equivalent to that of the parental mAb. In addition, nucleotide sequence comparison of 8C3 gene to the germline chicken V_L genes suggested that the gene conversion with V pseudogenes might contribute to the diversification of V_L genes in chickens.     

Eimeria acervulina: evaluation of the cellular and antibody responses to the recombinant coccidial antigens in B-congenic chickens

The roles of major histocompatibility complex (MHC) and non-MHC-linked genes in the genetic control of disease susceptibility and the development of protective immunity to Eimeria acervulina infection were investigated in six 15I5-B congenic and four different strains of chickens characterized for the MHC. When oocyst production was assessed, wide variations were noted following initial and challenge infections among the strains of chickens tested. In general, 15.N-21, 15.P-13, B21, B19, SC, and FP chickens were protected following challenge infection whereas 15I5, 15.P-19, 15.7-2, and 15.6-2 chickens were not. Strains of chickens sharing a same B haplotype on different genetic backgrounds did not show comparable levels of protection. These results lead to the view that non-MHC-linked genes have a profound influence on the outcome of the host response to E. acervulina infection. Chickens infected twice at 1-month intervals by an oral inoculation with E. acervulina developed both coccidial-specific antibody and T-cell responses. E. acervulina infected chickens showed T-cell-mediated immune responses to the intact sporozoites as well as to recombinant proteins, p130 of sporozoites and p150 of merozoites. Both p130 and p150 antigens have been identified and characterized previously. Sera obtained from all infected chickens recognized the p150 merozoite protein, but not the p130 sporozoite protein in immunoblots. In general, the cellular response, but not the antibody response to the p150 recombinant surface merozoite antigen correlated with the degree of protection following the challenge infection. These results suggest that the strains of chickens having improved protection against challenge infection demonstrate higher T-cell responses to the recombinant surface merozoite protein, p150.