Antibody Response In Chickens Research Articles

Crosstalk between innate and adaptive immune responses to infectious bronchitis virus after vaccination and challenge of chickens varying in serum mannose-binding lectin concentrations

Mannose-binding lectin (MBL), a C-type collectin with structural similarities to C1q, is an innate pattern-recognition molecule that is sequestered to sites of inflammation and infections. MBL selectively binds distinct chemical patterns, including carbohydrates expressed on all kinds of pathogens. The present study shows that serum MBL levels influence the ability of chickens to clear the respiratory tract of virus genomes after an infectious bronchitis virus (IBV) infection. The primary IBV infection induced changes in circulating T-cell populations and in the specific antibody responses. Serum MBL levels also influenced IBV vaccine-induced changes in circulating T-cell populations. Moreover, addition of mannose to an IBV vaccine altered both vaccine-induced changes in circulating T-cell populations and IBV specific vaccine and infection-induced antibody responses in chickens with high serum MBL levels. These data demonstrate that MBL is involved in the regulation of the adaptive immune response to IBV.

Evaluation of Salmonella enterica serovar Enteritidis pathogenicity island-1 proteins as vaccine candidates against S. Enteritidis challenge in chickens

Salmonella enterica serovar Enteritidis ( S. Enteritidis) is a major cause of gastrointestinal disease in humans worldwide, which mainly results from the consumption of contaminated poultry meat and eggs. Vaccination of chickens is an important strategy to lower the prevalence of Salmonella in poultry flocks. The S. Enteritidis type 3 secretion system (T3SS) encoded on Salmonella pathogenicity island-1 (SPI-1) is an important virulence factor that plays a role in invasion and systemic spread in chickens. In this manuscript, we evaluated the efficacy of SPI-1 proteins as vaccine candidates for protection against S. Enteritidis oral challenge. Our results demonstrate for the first time that SPI-1 T3SS proteins elicit antigen specific IgG antibody responses in chickens. In one study we show that vaccination with the aforementioned proteins reduces the levels of S. Enteritidis in the liver, but not in the spleen and cecal contents of chickens. However, a second study shows that vaccination of hens with SPI-1 proteins using a seeder model of infection does not affect the levels of S. Enteritidis in the cecal contents or internal organs of progeny obtained from these hens. Hence, the SPI-1 proteins, in conjunction with other proteins, may form important components of subunit vaccines used for protection against colonization by S. Enteritidis in poultry.

Differentiation of Infected and Vaccinated Animals (DIVA) Using the NS1 Protein of Avian Influenza Virus

Vaccination against avian influenza (AI) virus, a powerful tool for control of the disease, may result in issues related to surveillance programs and international trade of poultry and poultry products. The use of AI vaccination in poultry would have greater worldwide acceptance if a reliable test were available that clearly discriminated between naturally infected and vaccinated-only animals (DIVA). Because the nonstructural protein (NS1) is expressed in influenza virus-infected cells, and it is not packaged in the virion, it is an attractive candidate for a DIVA differential diagnostic test. The aim of this work was to determine the onset of the antibody response to the NS1 protein in chickens infected with low pathogenic avian influenza (LPAI) virus, and to evaluate the diagnostic potential of a baculovirus-expressed purified NS1 protein in an indirect ELISA-based DIVA strategy. An antibody response against NS1 was first detected 3 wk after infection, but the antibody levels were decreasing rapidly by 5 wk after infection. However, most chickens did not have detectable antibodies in spite of high hemagglutination inhibition (HI) antibody titers in one group. In birds vaccinated with inactivated oil-emulsion vaccines, antibodies against NS1 were not detected before virulent challenge, and only a small percentage of birds seroconverted after homologous LPAI virus challenge. Vaccinated birds challenged with highly pathogenic AI showed a higher NS1 antibody response, but at most only 40% of birds seroconverted against NS1 protein by 3 wk after challenge. Because of the variability of seroconversion and the duration of the antibody response in chickens, the NS1 protein DIVA strategy did not perform as well as expected, and if this strategy were to be used, it would require sampling a higher number of birds to compensate for the lower seroconversion rate.

A Comparison of the Antibody Responses between Specific Pathogen-Free and Commercial Layers Immunized with an Influenza Vaccine Prepared from Inactivated Non-Pathogenic H5N1 Virus by Single Shot

It is known that antibody responses in chickens against invading organisms or antigens are considerably different among different lines. Thus, an avian influenza vaccine was prepared from inactivated whole particles of the virus of non-pathogenic strain A/duck/Hokkaido/Vac-1/04 (H5N1) using an oil adjuvant containing anhydromannitol-octadecenoate-ether and injected intramuscularly into each ten 10-week-old specific pathogen-free (SPF) white leghorn chickens and commercial layers of Julia and Boris-Brown to obtain comparative data for antibody responses until 6 weeks after vaccination. Despite significant partial differences of antibody titer between the chicken lines, this study clearly showed that the vaccine induced good and sufficient antibody response in both SPF chickens and commercial layers.

Toward the Development of a Plant-Based Vaccine Against Reovirus

Transgenic lines of Arabidopsis thaliana producing recombinant TC protein were developed. The S1 gene encoding sigmaC protein of an avian reovirus (ARV) was amplified by reverse-transcription PCR (RT-PCR). The amplified product was cloned into a plant-expression vector, pE1857, with a strong promoter for expression. The resulting construct with the BAR gene cassette for bialaphos selection was designated rpE-sigmaC and was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-sigmaC constructs that transform A. thaliana and transgenic plants were selected using bialaphos selection. The presence of S1 transcript in plants and their relative level of expression were determined by real time RT-PCR. Western blot analysis further confirmed the presence of sigmaC protein in the plants. Transgenic lines with high levels of sigmaC protein were selected for immunization experiments using specific-pathogen free chickens. Recombinant sigmaC protein produced in plants induced a variety of immunoglobulin G (IgG) antibody responses in chickens. Recombinant protein administered either subcutaneously or orally in birds showed significant protection against challenge. Results suggested that the recombinant sigmaC protein produced in plants has the potential for large-scale vaccination against ARV in commercial poultry.

O.7.1 Gene expression analysis of cells infected with different human and avian influenza A virus strains

Avian influenza (AI) is a highly contagious disease in poultry and outbreaks can have dramatic economic and health implications. For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies against AI virus (AIV) proteins. In this study, we report the development of a multiplexed fluorescence microsphere immunoassay (FMIA) for detection of antibodies against AIV proteins in poultry. Recombinant nucleoprotein (NP), matrix protein (M1), and non-structural protein 1 (NS1) were expressed using a baculovirus expression system, purified and covalently coupled to fluorescent xMAP microspheres. Using these reagents, a triplex bead assay was developed for the Luminex platform. The assay displayed minimal cross reactivity when screened against a panel of reference sera raised against common avian viruses. For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commercially available ELISA. The assay was also employed to investigate the early kinetics of antibody response in chickens infected with AIV. Our results suggest that NP should be the protein of choice when detecting AI infections in commercial chickens, as the immune response was higher and persisted longer than that of M1 and NS1 proteins. This report provides a framework from which a more robust assay could be developed to profile exposure to many AIV subtypes in a single test.

A multiplexed immunoassay for detection of antibodies against avian influenza virus

Avian influenza (AI) is a highly contagious disease in poultry and outbreaks can have dramatic economic and health implications. For effective disease surveillance, rapid and sensitive assays are needed to detect antibodies against AI virus (AIV) proteins. In this study, we report the development of a multiplexed fluorescence microsphere immunoassay (FMIA) for detection of antibodies against AIV proteins in poultry. Recombinant nucleoprotein (NP), matrix protein (M1), and non-structural protein 1 (NS1) were expressed using a baculovirus expression system, purified and covalently coupled to fluorescent xMAP microspheres. Using these reagents, a triplex bead assay was developed for the Luminex platform. The assay displayed minimal cross reactivity when screened against a panel of reference sera raised against common avian viruses. For detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commercially available ELISA. The assay was also employed to investigate the early kinetics of antibody response in chickens infected with AIV. Our results suggest that NP should be the protein of choice when detecting AI infections in commercial chickens, as the immune response was higher and persisted longer than that of M1 and NS1 proteins. This report provides a framework from which a more robust assay could be developed to profile exposure to many AIV subtypes in a single test.

Modulation of the immune responses in chickens by low-pathogenicity avian influenza virus H9N2

Most low-pathogenicity avian influenza (LPAI) viruses cause no or mild disease in avian species. Little is known about the mechanisms of host defence and the immune responses of avian influenza-infected birds. This study showed that chicken macrophages are susceptible to infection with LPAI H9N2 and H6N2 viruses and that infection led to apoptosis. In H9N2 virus-infected chicken macrophages, Toll-like receptor 7 responded to infection and mediated the cytokine responses. Whilst pro-inflammatory cytokines were largely upregulated, the interferon (IFN) response was fairly weak and IFN-inducible genes were differentially regulated. Among the regulated genes, major histocompatibility complex (MHC) antigens II were downregulated, which also occurred in the lungs of H9N2-infected chickens. Additionally, interleukin (IL)-4, IL-4 receptor and CD74 (MHC class II invariable chain) were also downregulated, all of which are pivotal in the activation of CD4+ helper T cells and humoral immunity. Remarkably, in H9N2 virus-infected chickens, the antibody response was severely suppressed. This was in contrast to the robust antibody response in chickens infected with H6N2 virus, in which expression of MHC class II antigens was upregulated. These data suggest that neutralizing antibodies and humoral immunity may not be developed efficiently in H9N2-infected chickens. These findings raise questions about how some LPAI viruses differentially regulate avian immune responses and whether they have similar effects on mammalian immune function.

Antibody Response of Chickens to Serotype 1, 2, or 3 Marek's Disease Vaccines Based on ELISA with Infected Cells as Antigen

An enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the antibody response of commercial White Leghorn chickens to vaccination against Marek's disease (MD) at hatch (day 0) with serotype-1 (Rispens), -2 (SB-1), or -3 (turkey herpesvirus, HVT) vaccine virus and to challenge on day 21 with MD virus. Antigens for the test were whole chicken embryo fibroblast cells infected with Rispens, SB-1, or HVT. The chickens were progeny of stock that had been vaccinated with HVT, and on day 21 the nonvaccinated group had higher levels of maternal antibodies to HVT than to other antigens (P < 0.05). Only SB-1 vaccine had induced antibodies by day 21, and this was detected only against homologous antigens. On day 49, all three vaccines had induced higher levels of antibodies to homologous than to heterologous antigens. Marek's Disease virus (MDV) induced antibodies to all three antigens, but challenging vaccinated chicks did not significantly increase levels of antibodies on day 81 to any of the three antigens. It was concluded that an ELISA using whole cells as antigens would have potential value for monitoring the antibody response induced by MD vaccines and virulent MDV.

Effect of four adjuvants on immune response to F4 fimbriae in chickens

IgG antibody response in chickens immunized with F4 fimbriae extracted from local enterotoxigenic Escherichia coli (ETEC) strain was studied during a 98-day immunization period for comparing the efficacy of four adjuvants: Freund’ adjuvant, Quil A (QA), propolis and extract from Cochinchina momordica seed (ECMS). For this purpose, chickens were immunized with F4 fimbriae alone or combined with one of the above adjuvants on days 1 and 21. IgG antibody levels in serum and egg yolk (by ELISA) were measured on days 0, 7, 14, 21, 28, 35, 42, 49, 56, 70, 84 and 98. The egg production of each group was also determined during days 1–7 and the following four weeks. The results showed that QA could enhance antibody titre, as good or almost as good as Freund's adjuvant, whereas the titres of ECMS and propolis groups were relatively lower, with the overall order: Freund's adjuvant > QA > ECMS > propolis both in serum and egg yolk. However, the significant decrease of egg production was merely observed in the Freund's adjuvant group. It is concluded that the four adjuvants tested can stimulate immune response to F4 fimbriae in chickens, with Freund's adjuvant giving the best results, followed by QA.

Evaluation of Pathogenicity of Avian Poxvirus Isolates from Endangered Hawaiian Wild Birds in Chickens

Pathogenicity of two avian poxviruses isolated from endangered Hawaiian wild birds, the Hawaiian Goose and the Palila, was compared with fowl poxvirus in chickens. Immune responses were measured by ELISA pre- and postimmunization with Hawaiian poxviruses and after challenge with fowl poxvirus. Both isolates from Hawaiian birds developed only a localized lesion of short duration at the site of inoculation in specific-pathogen-free chickens and did not provide protection against subsequent challenge with virulent fowl poxvirus. On the other hand, birds inoculated with virulent fowl poxvirus developed severe lesions. In contrast to high antibody response in chickens immunized with fowl poxvirus, birds immunized with either of the two Hawaiian isolates developed low to moderate antibody responses against viral antigens. The level of immune responses, however, increased in birds of all groups following subsequent challenge.

Homotopes affect primary and secondary antibody responses in poultry

Activation of the innate immune system by different homotopes may direct the subsequent specific immune response, and as a consequence may have significant implications on vaccination. In this study, effects of i.v. administered lipopolysaccharide (LPS), and lipoteichoic acid (LTA), respectively, and s.c. administered Keyhole Limpet Hemocyanin (KLH) on total and isotype-specific (IgM and IgG) primary and secondary antibody responses of chickens to human serum albumin (HuSA) were determined. Similarly, effects of pre-treatment of the birds with the homotopes on primary antibody responses to rabbit gamma globulin (RGG) were measured. In addition, antigen-specific (HuSA and RGG) and mitogenic (concanavalin A, Con A) responses of peripheral blood leucocytes in vitro were also measured. Our findings confirm earlier observations that both LTA as well as LPS have immunomodulatory features in chickens, albeit in an opposite direction. LTA enhanced primary and to a minor degree secondary antigen-specific antibody titers, but LPS was found to suppress these responses. In RGG-primed birds, LPS enhanced a non-antigen-specific recall response to HuSA. In addition, KLH acted in an immunomodulatory role too, enhancing primary and secondary antibody responses, but suppressing non-specific cellular immunity in a non-antigen-specific fashion. Possible mechanisms underlying our observations, and the role of LTA, LPS, and KLH in polarization of the specific antibody response in chickens are discussed.

Associations of interferon-gamma genotype and protein level with antibody response kinetics in chickens.

Although previous studies have demonstrated an association between interferon-gamma (IFN-gamma) promoter genotype and antibody response kinetics in chickens, the protein levels that may mediate such a gene-trait association have not been determined. The objective of this study, therefore, was to determine the correlation of circulating IFN-gamma levels with both the IFN-gammaIFN-gamma promoter polymorphisms and antibody response in order to evaluate the potential role of IFN-gamma protein in mediating genetic control of antibody response in chickens. Antibody response after Salmonella enteritidis (SE) vaccination at day 10, antibody response to sheep red blood cells (SRBCs) and killed Brucella abortus after immunizations at 19 wk and 22 wk, and serum IFN-gamma protein level were measured in an F2 population derived from inbred lines. A single nucleotide polymorphism in the IFN-gamma promoter region was associated with IFN-gamma protein expression as measured by an enzyme-linked immunosorbent assay after both primary and secondary immunizations. Higher IFN-gamma protein level was correlated with higher antibody level to SE and with increased maximum level and decreased time to reach the maximum secondary antibody response to SRBCs. These results suggest that one of the mechanisms by which promoter polymorphism of IFN-gamma affects antibody production in chickens may involve the circulating level of IFN-gamma protein.

Different sources of dietary n-6 polyunsaturated fatty acids and their effects on antibody responses in chickens

1. Effects of linoleic and linolenic acid provided via different oil sources on total antibody (Ab) titres, Ab isotypes after primary and secondary immunisation, and cutaneous hypersensitivity (CH) responses to bovine serum albumin (BSA) and maleyl-BSA, respectively, were studied in pullets fed on one of 4 diets. The diets were the basal control diet enriched with either sunflower oil or safflower oil as sources of linoleic acid, and linseed oil as a source of linolenic acid, tested against a control diet supplemented with animal fat. 2. Total Ab and immunoglobulin (Ig) isotype responses to BSA were affected by diet after primary, and diet × immunisation effects after secondary immunisation. Higher total Ab and IgG titres to BSA were found especially after primary immunisation in birds given the sunflower oil enriched diet, whereas birds given sunflower oil mounted significantly lower IgM titres to BSA after primary and secondary immunisation. The antibody responses to maleyl-BSA were affected by diet after primary, and immunisation × diet interactions after secondary immunisation. Sunflower oil enhanced total and IgG Ab titres to maleyl-BSA after primary immunisation, but decreased IgM titres to maleyl-BSA after primary and secondary immunisation. Cutaneous hypersensitivity responses to BSA and maleyl-BSA were not affected by the diet. 3. It is concluded that modulation of the magnitude and isotype of Ab responses of poultry to T cell-dependent antigens is affected not only by type of essential fatty acids, but also by their source. In the present study the n-6 source, sunflower oil, showed strong enhancement of primary Ab responses, directed to both Th2 and Th1 antigens. On the other hand, the different effects of safflower oil imply that constituents other than n-6 acids within dietary plant oils may affect immune responsiveness. 4. The relationship between magnitude and isotype of Ab responsiveness, type of antigen, and essential fatty acids is discussed.

Initiation of humoral immunity. I. The role of cytokines and hormones in the initiation of humoral immunity using T-independent and T-dependent antigens

The early events during the initiation of immune responses following the injection of T-independent (lipopolysaccharide, LPS) and T-dependent (bovine serum albumin, BSA) antigens were studied in immature male chickens. Specifically, the role of cytokines and hormones in the initiation of humoral immunity against these antigens was investigated. Both interleukin-1 (IL-1) and tumor necrosis factor (TNF-α) increased significantly post-LPS but not post-BSA injection. While interleukin-2 (IL-2) significantly decreased post-LPS injection, IL-2 significantly increased post-BSA injection. Furthermore, corticosterone levels significantly increased and tri-iodothyronine (T 3) levels significantly decreased post-LPS but not post-BSA injection. In this study, the results indicate that although LPS and BSA can induce a humoral antibody response in chickens, they activate different cytokines and neuroendocrine network systems.

Localization and immunogenicity of a low molecular weight antigen of Eimeria tenella.

A low molecular weight (LMW) antigen recognized by a murine monoclonal antibody (C(3)4F(1)) was localized within endogenous stages of Eimeria tenella (USDA strain 80). Using indirect fluorescent antibody assay and immunoelectron microscopy, the LMW antigen was found in: sporozoites, first, second and third generation meronts, gamonts, unsporulated oocysts, and sporocysts. The antigen was observed in the cytoplasm and pellicle of the parasite, and in the parasitophorous vacuole, sporocyst walls and cytoplasm of infected host cells. The immunogenicity of this LMW antigen was assessed by antigen-specific serum antibody responses in chickens orally inoculated with live oocysts or injected intramuscularly with dead sporozoites. LMW antigen-specific serum antibodies were detected using Western blots of E. tenella sporozoites as early as 4 days after sporozoite injection and 6 days after oocyst inoculation. Unusually, the monoclonal antibody C(3)4F(1) reduced the binding of immune chicken serum to the antigen in a competitive antibody binding assay, but not the reverse, suggesting that there is a single, immunodominant epitope on this antigen.

Major histocompatibility complex (MHC) related cDNA probes associated with antibody response in meat-type chickens.

The major histocompatibility complex (MHC) region was examined as a set of candidate genes for association between DNA markers and antibody response. Intercross F2 families of chickens were generated from a cross between high (HC) and low (LC) Escherichia coli(i) antibody lines. Restriction fragment length polymorphism (RFLP) analysis was conducted by using three MHC-related cDNA probes: chicken MHC class IV (B-G), chicken MHC class I (B-F), and human MHC-linked Tap2. Association between RFLP bands and three antibody response traits (E. coli, sheep red blood cells and Newcastle disease virus) were determined by two methods: by statistically analyzing each band separately and also by analyzing all bands obtained from the three probes by using multiple regression analysis to account for the multiple comparisons. The MHC class IV probe was the highest in polymorphisms but had the lowest number of bands associated with antibody response. The MHC class I probe yielded 15 polymorphic bands of which four exhibited association with antibody response traits. The Tap2 probe yielded 20 different RFLP bands of which five were associated with antibody production. Some Tap2 bands were associated with multiple antibody response traits. The multiband analysis of the three probes' bands revealed more significant effects than the analysis of each band separately. This study illustrates the efficacy of using multiple MHC region probes as candidate markers for quantitative trait loci (QTLs) controlling antibody response in chickens.

Comparison of adjuvants with respect to serum IgG antibody response in orally immunized chickens

We have previously shown that oral immunization with non-replicating antigens hardly induced serum IgG antibody response in chickens and addition of sodium fluoride (NaF) to the immunogen markedly improved their immunological states. In the present study, taurine, lithium and Quillaja saponin ( Q-SAP) were compared with NaF with respect to their enhancement of serum IgG antibody response in chickens after oral immunization. The antibody titer of chickens which received Q-SAP as the mucosal adjuvant tended to be higher than that of chickens which received antigen plus NaF. Simultaneous administration of antigen with lithium or taurine elicited a higher antibody titer in chickens compared to those of chickens orally immunized with antigen alone, but the effect of these two adjuvants was less efficient compared with that of NaF. These results suggested that Q-SAP as well as NaF is useful as an oral adjuvant for chickens.

Coadministration of IFN-gamma enhances antibody responses in chickens.

The development of new generation vaccines has focused on the use of natural immunologic adjuvants that are capable of enhancing a protective immune response. The use of cytokines as immunomodulators in livestock animals, particularly poultry, is becoming more feasible with the recent cloning of several cytokine genes and the progression of new delivery technologies, such as live vectors and DNA delivery. Given that chickens are reared under intensive conditions that are conducive to infection by opportunistic pathogens, the primary mechanism for disease control in poultry is early and effective vaccination. However, many poultry vaccines offer only short-term protection or give nonuniform responses within flocks. We have developed a model system with which to measure the adjuvant potential of cytokines in chickens. This involves measuring antibody levels following coadministration of chicken interferon-gamma (Ch-IFN-gamma) with sheep red blood cells (SRBC). Groups of SPF and commercial broiler birds were injected with two different doses of SRBC with and without coadministration of Ch-IFN-y. Three weeks later, all birds were boosted with SRBC alone. Sera were collected weekly and anti-SRBC antibody titers (total Ig and IgG) were determined by hemagglutination. Priming Ch-IFN-gamma resulted in enhanced primary and secondary (IgG) antibody responses that persisted at higher levels when compared with birds that received SRBC alone. Second, coadministration of Ch-IFN-y allowed a 10-fold lower dose of antigen to be as effective as a high dose of antigen that was given without Ch-IFN-gamma. Third, treatment with Ch-IFN-y resulted in an increase in the proportion of birds responding to antigen challenge. These results suggest the potential use for Ch-IFN-gamma as a vaccine adjuvant.

Influence of inoculation site of combined oil-adjuvanted vaccine on the antibody response in chickens.

Inactivated oil-adjuvanted vaccines for ND, IB, and IC serotypes A and C (OILVAX NB2AC) have been marketed from 1993. In the outdoors, various inoculation sites have been used in chickens because to make the inoculation procedure easier. We examined whether differences would be obtained in the antibody response according to the inoculation sites; the subcutaneous inoculation into the back of the neck for OILVAX use, and the thigh, lower thigh, breast and shoulder muscle for the possible application of the inoculation outdoors. The clear order was not found in the NDV-HI titer, IBV-SN titer against Nerima or TM-86 among the inoculation sites used during the examination period. The IC serotypes A and C HI titers did vary among the inoculation sites; the subcutaneous inoculation produced the highest antibody titer, and high antibody titers were observed in the order of lower thigh muscle > or = thigh muscle > breast muscle > or = shoulder muscle inoculation.