Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck

Galliform and non-galliform birds express three immunoglobulin isotypes, IgM, IgA and IgY. Beyond this we should not generalize because differences in gene organization may have functional consequences reflected in the immune response. At present, studies on non-galliform birds are largely restricted to ducks. Ducks express an alternatively spliced form of their IgY heavy chain (upsilon) gene, the IgY(DeltaFc), that lacks the Fc region and Fc-associated secondary effector functions. It is not known how common the expression of the IgY(DeltaFc) is among birds, nor the functional consequences. It is also not known whether the unusual organization of the duck IgH locus, also shared with the chicken, having the gene order of mu, alpha and upsilon, with alpha inverted in the locus, is unique to the galloanseriform lineage. Ducks, like chickens, have a single immunoglobulin light chain of the lambda (lambda) type. Evidence suggests that ducks, like chickens, generate their immunoglobulin repertoire through a single functional rearrangement of the variable (V) region, and generate diversity through gene conversion from a pool of pseudogenes. In Southern blots of germline and rearranged bursal DNA, both the heavy and light chain loci of ducks appear to each undergo one major rearrangement event. For both heavy and light chains, the functional V region element and the pseudogenes appear to consist of a single gene family. Further analysis of 26 heavy chain joining (JH) and 27 light chain JL segments shows there is use of a single J segment in ducks, which is diversified presumably through somatic mutations and gene conversion events. Despite this limitation on the rearrangement of immunoglobulin genes, analysis of 26 DH and 122 VL sequences suggests that extensive sequence diversity is generated.     

Defense genes missing from the flight division

Birds have a smaller repertoire of immune genes than mammals. In our efforts to study antiviral responses to influenza in avian hosts, we have noted key genes that appear to be missing. As a result, we speculate that birds have impaired detection of viruses and intracellular pathogens. Birds are missing TLR8, a detector for single-stranded RNA. Chickens also lack RIG-I, the intracellular detector for single-stranded viral RNA. Riplet, an activator for RIG-I, is also missing in chickens. IRF3, the nuclear activator of interferon-beta in the RIG-I pathway is missing in birds. Downstream of interferon (IFN) signaling, some of the antiviral effectors are missing, including ISG15, and ISG54 and ISG56 (IFITs). Birds have only three antibody isotypes and IgD is missing. Ducks, but not chickens, make an unusual truncated IgY antibody that is missing the Fc fragment. Chickens have an expanded family of LILR leukocyte receptor genes, called CHIR genes, with hundreds of members, including several that encode IgY Fc receptors. Intriguingly, LILR homologues appear to be missing in ducks, including these IgY Fc receptors. The truncated IgY in ducks, and the duplicated IgY receptor genes in chickens may both have resulted from selective pressure by a pathogen on IgY FcR interactions. Birds have a minimal MHC, and the TAP transport and presentation of peptides on MHC class I is constrained, limiting function. Perhaps removing some constraint, ducks appear to lack tapasin, a chaperone involved in loading peptides on MHC class I. Finally, the absence of lymphotoxin-alpha and beta may account for the observed lack of lymph nodes in birds. As illustrated by these examples, the picture that emerges is some impairment of immune response to viruses in birds, either a cause or consequence of the host-pathogen arms race and long evolutionary relationship of birds and RNA viruses.     

The ratio of full length IgY to truncated IgY in immune complexes affects macrophage phagocytosis and the acute phase response of mallard ducks (Anas platyrhynchos)

Ducks produce a full length IgY and a truncated isoform (IgYDeltaFc). IgY and IgY(DeltaFc) were isolated from ducks vaccinated against Escherichia coli and used to make E.coli-Ig immune complexes (IC). Phagocytosis of IC by duck monocytes decreased directly with the proportion of IgYDeltaFc (p<0.001). IC containing IgY:IgY(DeltaFc) at ratios of 100:0, 50:50, 0:100, and 0:0 (E. coli alone) were injected intravenously into na?ve mallard ducks. At 24 h after injection, plasma hemopexin levels were higher in ducks given either the 0:100 ratio or the 100:0 ratio than those given 50:50 or E. coli alone (p<0.005) Liver IL-1beta mRNA levels followed a similar pattern. Splenic IL-1beta mRNA decreased markedly as the proportion of IgY(DeltaFc) increased (p<0.01) Thus, IgY(DeltaFc) may shift the response to IC from the spleen to the liver as infections progress from acute to chronic.     

Duck immunoglobulins: structure, functions and molecular genetics

Four types of immunoglobulin (Ig) have been identified in ducks: IgM, a secretory Ig resembling IgM, a 7.8S IgG, and a 5.7S IgG. Structurally and antigenically the 5.7S IgG resembles an F(ab')2 fragment of the 7.8S IgG. When ducks mount serum antibody responses, the sequence of Ig involvement is IgM ? 7.8S IgG ? 5.7S IgG. Although serum Ig levels increase, and antigen‐binding Igs can be demonstrated, sera from repeatedly immunized ducks commonly lack secondary antibody activities such as agglutination, precipitation, complement fixation and tissue sensitization. These deficiencies are most likely attributable to the absence of functionally important components of the predominant Ig (5.7S IgG) and/or a possibly unusual steric structure of duck Igs. A related issue concerns production of the two antigenically related IgGs: what are the cellular and molecular events involved, and how are they controlled?

Evidence from current molecular genetic studies has confirmed the similarity of the V_H, C_H1 and Ch2 domains of the 7.8S and 5.7S IgGs and shown, by virtue of the existence of separate mature messages for the heavy (H) chains of these molecules, that they are biosynthesized independently. Models for the possibilities that the two H chains are products of one gene or of two genes are presented. Cloning and sequencing the duck H chain gene locus, which is in progress, is providing data supporting the one gene hypothesis. The results obtained from cDNA sequencing also confirm that the duck IgGs are unusual in terms of the anatomy of the hinge region and of the number and location of intra‐ and inter‐chain disulphide bonds, observations which will be of importance for understanding structure/function relationships of these unusual and interesting molecules.     

Evaluation of Newcastle disease virus immunoassays for waterfowl using a monoclonal antibody specific for the duck immunoglobulin light chain

In the present study a monoclonal antibody (mAb 14A3) was tested for its reactivity against serum immunoglobulin Y (IgY) of several waterfowl species, and subsequently for its applicability as anti-species antibody in common immunoassays. Western blot analyses demonstrated its broad cross-reactivity with the serum IgY light chain of different duck species: Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), white-winged wood duck (Asarcornis scutulatus), common pintail (Dafila acuta). Reactivity was also evident with IgY of two swan species—mute swan (Cygnus olor) and black-necked swan (Sthenelides melanocoryphus)—and two goose species—domestic goose (Anser anser var. domestica) and red-breasted goose (Rufibrenta ruficollis). Applying the mAb for Newcastle disease virus (avian paramyxovirus serotype 1 [APMV-1]) test systems, its functionality within indirect immunoassays was evaluated. Using APMV-1-positive sera of domestic geese and Muscovy ducks, mAb 14A3 facilitated specific staining of APMV-1-infected cells in an immunofluorescence test. In addition, it proved to be functional in an indirect enzyme-linked immunosorbent assay (ELISA) and a western blot assay. Thus, the analysed mAb represents an attractive and versatile reagent that offers the opportunity to develop serological tests for waterfowl, allowing a high sample throughput using the ELISA technique or the fine analysis of humoral immune responses using the western blot.     

Differential innate responses of chickens and ducks to low-pathogenic avian influenza

Ducks and chickens are hosts of avian influenza virus, each with distinctive responses to infection. To understand these differences, we characterized the innate immune response to low-pathogenicity avian influenza virus H7N1 infection in chickens and ducks. Viral RNA was detected in the lungs of chickens from day 0.8 to 7, in ducks mainly at day 4. In both species, viral RNA was detected in the bursa and gut. Infection in chickens resulted in up-regulation of interferon (IFN)-α and IFN-β mRNA, while in the ducks IFN-γ mRNA was strongly up-regulated in the lung and bursa. In chickens and ducks, all investigated pathogen recognition receptor (PRR) mRNAs were up-regulated; however, in the chicken lung Toll-like receptor (TLR)7 and melanoma differentiation-associated protein (MDA)-5 mRNA were strongly induced. TLR3, TLR7 and MDA-5 responses correlated with IFN-α and IFN-β responses in chickens, but in ducks a correlation between IFN-α and TLR7, retinoic acid-inducible gene-I and MDA-5 was absent. We studied the responses of duck and chicken splenocytes to poly(I:C) and R848 analogues to analyse the regulation of PRRs without the interfering mechanisms of the influenza virus. This revealed IFN-α and IFN-γ responses in both species. MDA-5 was only strongly up-regulated in chicken splenocytes, in which time-related PRR responses correlated with the IFN-α and IFN-β response. This correlation was absent in duck splenocytes. In conclusion, chickens and ducks differ in induction of MDA-5, TLR7 and IFN-α mRNA after an influenza virus infection in vivo and after in vitro stimulation with TLR antagonists.     

Studies with inactivated duck virus hepatitis vaccines in breeder ducks

Inactivated duck virus hepatitis (DVH) oil emulsion vaccines were prepared and evaluated in White Pekin breeder ducks and compared to a live DVH vaccine obtained from a commercial source. The response to vaccination was measured by serum neutralisation (SN) tests and resistance to experimental challenge in ducklings bred from vaccinated parents 32-35 weeks of age. Inactivated vaccines prepared from DVH virus grown in duck eggs stimulated a better antibody response than vaccines prepared from virus grown in chicken eggs. This was probably due to the higher yield of virus obtained from the duck eggs. Neither inactivated vaccine produced a satisfactory antibody response when used as a primary vaccine in 16-week-old ducks. Using the inactivated vaccine prepared from virus grown in duck eggs an improved antibody response was recorded when the vaccine was given in three doses at 8, 16 and 22 weeks of age. The levels of antibody in the breeding ducks was sufficiently high to confer protection to their progeny for up to 3 weeks of age. Ducks vaccinated at 2-3 days of age with the live DVH vaccine produced a high and sustained antibody response for up to 30 weeks. Ducklings bred from this group were resistant to challenge for up to 3 weeks of age. Ducks that were given live DVH vaccine at 2-3 days of age followed by inactivated vaccine at 22 weeks of age produced SN antibody levels significantly higher than levels produced from three doses of inactivated vaccine or one dose of live vaccine. Ducklings bred from these ducks were resistant to challenge for up to 3 weeks of age. These results suggest that inactivated DVH vaccines may be of value where the use of live DVH vaccines are contra-indicated.     

Duck influenza lacking evidence of disease signs and immune response.

Influenza viruses A/duck/Hokkaido/5/77 (Hav7N2), A/budgerigar/Hokkaido/1/77 (Hav4Nav1), A/Kumamoto/22/76 (H3N2), A/Aichi/2/68 (H3N2), and A/New Jersey/8/76 (Hsw1N1) were experimentally inoculated into Pekin ducks. Of these, the influenza viruses of duck and budgerigar origin replicated in the intestinal tract of the ducks. The infected ducks shed the virus in the feces to high titers, but did not show clinical signs of disease and scarcely produced detectable serum antibodies. Using immunofluorescent staining, we demonstrated that the target cells of the duck virus in ducks were the simple columnar epithelial cells which form crypts in the large intestines, especially in the colon. After primary infection, the birds resisted reinfection with the duck virus at least for 28 days, but from 46 days onward they were susceptible to reinfection. These infections were quickly restricted by a brisk secondary immune response, reflected in the rapid appearance of high titers of antibody after reinoculation. In contrat to the avian influenza viruses, the remaining three influenza viruses of human origin did not replicate in the intestinal tract but did cause a serum antibody response.     

Duck antibody responses to keyhole limpet haemocyanin,human immunoglobulin G and the trinitrophenyl hapten. Evidence of affinity maturation

White Pekin ducks (three in each of four groups) received trinitrofluorobenzene (TNP) conjugated to keyhole limpet haemocyanin (KLH) or human immunoglobulin G (HIgG) either incorporated into adjuvants (complete Freund's adjuvant (CFA), then incomplete Freund's adjuvant (IFA) followed by intravenous (i.v.) injection) or by repeated i.v. injection. A rabbit received TNP-KLH with CFA, IFA, then i.v. Antibody (Ab) responses to the carrier proteins and to the TNP hapten were monitored by enzyme-linked immunosorbent assay (ELISA), optimized for use with duck Abs, and the response to TNP was also assessed in an ELISA designed to detect shifts in Ab affinity (Ka). The rabbit mounted good Ab responses to KLH and TNP, the Ka of the anti-TNP response increasing from 10^5.99 to 10^8.87 l/mol (758-fold) during the experiment. Ducks showed weak Ab responses to KLH and HIgG, with weakest responses among ducks receiving antigen by the i.v. route exclusively. The duck anti-TNP responses were vigorous in all cases. However, following i.v. administration of antigens (Ags), the anti-TNP responses were transient, being strong by 7 days but diminishing to background levels by 14 to 28 days after each inoculation. The duck Ab responses to TNP displayed affinity maturation. In ducks receiving Ags with adjuvants, Ka increased by levels comparable with or even exceeding those in the rabbit. Affinity increases were less, but nevertheless apparent, following i.v. administration of Ag. These results show that, contrary to expectation, the duck Ab response experiences affinity maturation. They also point to the transience of the duck Ab response to selected haptens/epitopes when Ag is not delivered in adjuvant. These findings have clinical implications for field responses to pathogens and vaccines.     

Cloning,expression and purification of duck hepatitis B virus (DHBV) core protein and its use in the development of an indirect ELISA for serologic detection of DHBV infection

Infecting ducks with duck hepatitis B virus (DHBV) is widely accepted as a relevant model for studying aspects of human HBV infection. However, efficient and sensitive diagnostic methods for the various infection models are limited. In order to provide a more simple and convenient method for serologic diagnosis, we improved the production of recombinant DHBV viral capsid protein (core protein) and then used it to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detecting anti-DHBc antibodies (DHBcAg ELISA) in DHBV-infected ducks. Given the positive/negative cut-off value, the maximum dilution of duck sera in which anti-DHBc antibodies could be detected was 1:12,800. In addition, the DHBcAg ELISA displayed no cross reactivity with duck antisera against duck circovirus (DuCV), duck plague virus (DPV), duck hepatitis virus (DHV), duck swollen head septicemia virus (DSHSV), avian influenza virus (AIV), Riemerella anatipestifer, Salmonella anatum, or Escherichia coli. Furthermore, the coefficients of variation (CVs) of inter-assay and intra-assay experiments were both below than 10 %. When compared to PCR for accuracy on clinical samples from cases of suspected DHBV infection, the DHBcAg showed 95.45 % coincidence with PCR. In conclusion, recombinant DHBc was readily produced and used to establish a simple DHBcAg ELISA that provided a highly specific and sensitive method for analysis of clinical samples.     

Enhancement of the IgG antibody production to Escherichia coli O antigen by prior exposure to serologically different E. coli bacteria

Exposure of rabbits and mice to serologically different E. coli bacteria enhanced the synthesis of IgG but often suppressed the formation of IgM O antibodies in response to subsequent immunization with E. coli O6 bacteria. The effect differed with the O antigen used. A higher enhancing effect on the IgG antibody synthesis was associated with less suppression of the IgM-antibody formation. The antibody avidity of both immunoglobulin classes was relatively low compared to that in the ordinary immune response to E. coli O6. The findings indicated an enhancing action of endotoxin on the IgG antibody response to the O6 antigen.     

Dynamic gene expression analysis in a H1N1 influenza virus mouse pneumonia model

H1N1, a major pathogenic subtype of influenza A virus, causes a respiratory infection in humans and livestock that can range from a mild infection to more severe pneumonia associated with acute respiratory distress syndrome. Understanding the dynamic changes in the genome and the related functional changes induced by H1N1 influenza virus infection is essential to elucidating the pathogenesis of this virus and thereby determining strategies to prevent future outbreaks. In this study, we filtered the significantly expressed genes in mouse pneumonia using mRNA microarray analysis. Using STC analysis, seven significant gene clusters were revealed, and using STC-GO analysis, we explored the significant functions of these seven gene clusters. The results revealed GOs related to H1N1 virus-induced inflammatory and immune functions, including innate immune response, inflammatory response, specific immune response, and cellular response to interferon-beta. Furthermore, the dynamic regulation relationships of the key genes in mouse pneumonia were revealed by dynamic gene network analysis, and the most important genes were filtered, including Dhx58, Cxcl10, Cxcl11, Zbp1, Ifit1, Ifih1, Trim25, Mx2, Oas2, Cd274, Irgm1, and Irf7. These results suggested that during mouse pneumonia, changes in the expression of gene clusters and the complex interactions among genes lead to significant changes in function. Dynamic gene expression analysis revealed key genes that performed important functions. These results are a prelude to advancements in mouse H1N1 influenza virus infection biology, as well as the use of mice as a model organism for human H1N1 influenza virus infection studies.     

Immunotargeting with CD154 (CD40 ligand) enhances DNA vaccine responses in ducks

Engagement of CD154 on activated T cells with CD40 on antigen-presenting cells (APCs) potentiates adaptive immune responses in mammals. Soluble multimeric forms of CD154 have been used as an adjuvant or in immunotargeting strategies to enhance vaccine responses. The objective of our study was to examine the ability of duck CD154 (DuCD154) to enhance DNA vaccine responses in the duck hepatitis B model. Constructs were generated to express the functional domain of DuCD154 (tCD154), truncated duck hepatitis B virus (DHBV) core antigen (tcore) and chimera of tcore fused to tCD154 (tcore-tCD154). Expression in LMH cells demonstrated that all proteins were secreted and that tCD154 and tcore-tCD154 formed multimers. Ducks immunized with the plasmid ptcore-tCD154 developed accelerated and enhanced core-specific antibody responses compared to ducks immunized with ptcore or ptcore plus ptCD154. Antibody responses were better sustained in both ptcore-tCD154- and ptcore plus ptCD154-immunized ducks. Core-specific proliferative responses of duck peripheral blood mononuclear cells were enhanced in ducks immunized with ptcore-tCD154 or ptcore alone. This study suggests that the role of CD154 in the regulation of adaptive immune responses had already evolved before the divergence of birds and mammals. Thus, targeting of antigens to APCs with CD154 is an effective strategy to enhance DNA vaccine responses not only in mammalian species but also in avian species.     

Immunoglobulin G subclass levels and antibody responses to the 2009 influenza A (H1N1) monovalent vaccine among human immunodeficiency virus (HIV)-infected and HIV-uninfected adults

Immunoglobulin (Ig)G levels are important for antibody vaccine responses and IgG subclass deficiencies have been associated with severe 2009 influenza A (H1N1) infections. Studies have demonstrated variations in immune responses to the H1N1 vaccine, but the aetiology of this is unknown. We determined the associations between pre‐vaccination overall and influenza‐specific IgG subclass levels and 2009 H1N1‐specific antibody responses post‐vaccination (robust versus poor at day 28) stratified by human immunodeficiency virus (HIV) status. Logistic regression models were utilized to evaluate whether pre‐vaccination IgG subclass levels were associated with the antibody response generated post‐vaccination. We evaluated 48 participants as part of a clinical study who were stratified by robust versus poor post‐vaccination immune responses. Participants had a median age of 35 years; 92% were male and 44% were Caucasian. HIV‐infected adults had a median CD4 count of 669 cells/mm³, and 79% were receiving highly active anti‐retroviral therapy. HIV‐infected participants were more likely to have IgG2 deficiency (<240 mg/dl) than HIV‐uninfected individuals (62% versus 4%, P < 0·001). No association of pre‐vaccination IgG subclass levels (total or influenza‐specific) and the antibody response generated by HIN1 vaccination in either group was found. In summary, pre‐vaccination IgG subclass levels did not correlate with the ability to develop robust antibody responses to the 2009 influenza A (H1N1) monovalent vaccine. IgG2 deficiencies were common among HIV‐infected individuals but did not correlate with poor influenza vaccine responses. Further investigations into the aetiology of disparate vaccine responses are needed.     

Chronic Active Hepatitis Induced by Helicobacter hepaticus in the A/JCr Mouse Is Associated with a Th1 Cell-Mediated Immune Response

Helicobacter hepaticus infection in A/JCr mice results in chronic active hepatitis characterized by perivascular, periportal, and parenchymal infiltrates of mononuclear and polymorphonuclear cells. This study examined the development of hepatitis and the immune response of A/JCr mice to H. hepaticus infection. The humoral and cell-mediated T helper immune response was profiled by measuring the postinfection (p.i.) antibody response in serum, feces, and bile and by the production of cytokines and proliferative responses by splenic mononuclear cells to H. hepaticus antigens. Secretory immunoglobulin A (IgA) and systemic IgG2a antibody developed by 4 weeks p.i. and persisted through 12 months. Splenocytes from infected mice proliferated and produced more gamma interferon (IFN-γ) than interleukin-4 (IL-4) or IL-5 when cultured with H. hepaticus outer membrane proteins. The predominantly IgG2a antibody response in serum and the in vitro production of IFN-γ in excess of IL-4 or IL-5 are consistent with a Th1 immune response reported in humans and mice infected with Helicobacter pylori and Helicobacter felis, respectively. Mice infected with H. hepaticus developed progressively severe perivascular, periportal, and hepatic parenchymal lesions consisting of lymphohistiocytic and plasmacytic cellular infiltrates. In addition, transmural typhlitis was observed at 12 months p.i. The characterization of a cell-mediated Th1 immune response to H. hepaticus infection in the A/JCr mouse should prove valuable as a model for experimental regimens which manipulate the host response to Helicobacter.     

Humoral response of chicken infected with the microsporidium Encephalitozoon hellem

Chicken (Gallus gallus) were used as the experimental model for study of immune response against the microsporidium Encephalitozoon hellem (Didier et al., J Inf Dis 163:617–621, 1991) infection in birds. Two-day-old chicken were infected perorally or intraperitoneally with a dose of 10⁷ spores of E. hellem. The anti-E. hellem immunoglobulin (Ig)A, IgY, and IgM antibody responses in sera and dropping sample extracts were determined by enzyme-linked immunosorbent assay. Results have shown specific antibody production in sera and intestinal secretions of infected birds. Chicken inoculated perorally developed the lowest antibody response. Microsporidian spores were not identified in the smears from cloacal swab samples of individual chicken. Intestinal segment cultures of perorally infected chicken cultivated in vitro showed the highest production of specific IgY and IgA antibodies in jejunum segments. In the further course of infection, the colon produced the highest amount of IgA, and the ileum and colon produced the highest amount of IgY.