Liselotte R. Norup

Serum levels of mannan-binding lectin in chickens prior to and during experimental infection with avian infectious bronchitis virus

ABSTRACT Mannan-binding lectin (MBL) is a glycoprotein and a member of the C-type lectin super family, the collectin family, and the acute phase protein family. The MBL exerts its function by directly binding to microbial surfaces through its carbohydrate recognition domains, followed by direct opsonization or complement activation via MBL-associated serine proteases (MASP)-1 and -2. Thus, MBL plays a major role in the first-line innate defense against pathogens. We investigated the MBL concentrations in serum during experimental infectious bronchitis virus (IBV) infections in chickens. The results showed that the acute phase MBL response to infection with IBV was, to a degree (P < 0.0068), dependent on whether the chickens were inoculated after 12 h of rest (dark) or after 12 h of activity (light). The acute phase response in chickens challenged after 12 h of activity peaked after 4.6 d with an increase of 24%, whereas the acute phase response in chickens challenged after 12 h of rest peaked after 3.1 d with an increase of 51%. The specific antibody titer against IBV was also tested, and a difference (P < 0.0091) between the two experimental groups was found with peak titer values of 6,816 and 4,349. However, the highest value was found in chickens inoculated after 12 h of activity. Thus, an inverse relation exists between the MBL response and the IBV specific antibody response. The ability of MBL to activate the complement cascade was tested in a heterologous system by deposition of human C4 on the chicken MBL/MASP complex. The complement activation was directly associated with the concentration of MBL in serum, indicating neutralization of the virus before the humoral antibody response took over.

Flow cytometric assessment of chicken T cell-mediated immune responses after Newcastle disease virus vaccination and challenge.

The objective of this study was to use flow cytometry to assess chicken T cell-mediated immune responses. In this study two inbred genetic chicken lines (L130 and L133) were subjected to two times vaccination against Newcastle disease (ND) and a subsequent challenge by ND virus (NDV) infection. Despite a delayed NDV-specific antibody response to vaccination, L133 appeared to be better protected than L130 in the subsequent infection challenge as determined by the presence of viral genomes. Peripheral blood was analyzed by flow cytometry and responses in vaccinated/challenged birds were studied by 5-color immunophenotyping as well as by measuring the proliferative capacity of NDV-specific T cells after recall stimulation. Immunophenotyping identified L133 as having a significantly lower CD4/CD8 ratio and a lower frequency of gammadelta T cells than L130 in the peripheral T cell compartment. Furthermore, peripheral lymphocytes from L133 exhibited a significantly higher expression of CD44 and CD45 throughout the experiment. Interestingly, also vaccine-induced differences were observed in L133 as immune chickens had a significantly higher CD45 expression on their lymphocytes than the naïve controls. Immune chickens from both lines had a significantly higher frequency of circulating gammadelta T cells than the naïve controls both after vaccination and challenge. Finally, the proliferative capacity of peripheral CD4+ and CD8+ cells specific for NDV was addressed 3 weeks after vaccination and 1 week after infection and found to be significantly higher in L133 than in L130 at both sampling times. In conclusion, we found the applied flow cytometric methods very useful for the study of chicken T cell biology.

Influence of chicken serum mannose-binding lectin levels on the immune response towards Escherichia coli

This study aimed to investigate the effect of mannose-binding lectin (MBL) on infections with Escherichia coli in chickens. Initially, the basic levels of MBL in 4 different lines of layer chickens, namely ISA Brown, Lohmann Selected Leghorn, Lohmann Braun, and Hellevad, were investigated. This investigation revealed a 2-to 3-fold difference in the basic levels of MBL in serum between some of these commercial lines. Furthermore, the ontogeny of the basic level of MBL in serum of an experimental chicken line was investigated. The level of MBL was very stabile for long periods, with an elevation at 5 to 7 wk of age. Another elevation in MBL level started around 18 to 19 wk of age and stayed elevated at least until 38 wk of age. In this study, it was hypothesized that chickens with high levels of MBL (H-type) may be less prone to disease caused by E. coli infection than chickens with low levels of MBL (L-type) after attempts were made to immunosuppress the chickens by immunization with a live attenuated infectious bursal disease virus (IBDV) vaccine strain. The H-type and L-type chickens were divided into 4 groups receiving either no treatment (I-E-), E. coli alone (I-E+), IBDV alone (I+E-), or IBDV and E. coli (I+E+). Body weight gain was depressed by IBDV immunization as well as E. coli inoculation. The depression of BW gain was significantly larger in L-type chickens compared with H-type chickens. The antibody response to E. coli was significantly depressed by IBDV vaccination and antibody titers to E. coli were elevated by experimental E. coli inoculation, but only in the group not given IBDV (I-E- vs. I-E+). On d 28, T-cell responses in L-type chickens showed a lower percentage of proliferating CD4+ and CD8+ T cells compared with the H-type, regardless of treatment. In conclusion, immune reactions toward infections with E. coli differed between chickens having different basal serum MBL levels, and as such, MBL may be of importance for future selection of more robust chickens for outdoor or organic farming.

Comparison of parasite-specific immunoglobulin levels in two chicken lines during sustained infection with Ascaridia galli.

Increasingly large numbers of poultry are held in production systems with access to outdoor areas. In these systems intestinal helminths are found with flock prevalences of up to 100%. Helminth infections influence chicken health negatively, which is why the following investigation has been performed. In the present experiment, 20 chickens of two inbred chicken lines containing the major histocompatibility complex (MHC) haplotypes, B14 and R5, were inoculated with 500 embryonated Ascaridia galli eggs. The A. galli-specific IgG titres of serum samples and the excretion of A. galli eggs in chicken faeces were measured for a period of 81 weeks. The level of excreted A. galli eggs measured as eggs per gram chicken faeces (EPG) varied greatly between chickens in each line. Significant differences were found between the two lines and with the R5 chickens reaching the highest levels. Likewise, the A. galli-specific IgG titres in serum differed significantly between the two lines, and an inverse relationship between infection level (EPG) and antibody titres was found. Although this inverse relationship suggests that humoral immunity may be involved in protection against A. galli infection, the high antibody titres did not prevent continued infection.

Ascaridia galli infection influences the development of both humoral and cell-mediated immunity after Newcastle Disease vaccination in chickens.

Potent vaccine efficiency is crucial for disease control in both human and livestock vaccination programmes. Free range chickens and chickens with access to outdoor areas have a high risk of infection with parasites including Ascaridia galli, a gastrointestinal nematode with a potential influence on the immunological response to vaccination against other infectious diseases. The purpose of this study was to investigate whether A. galli infection influences vaccine-induced immunity to Newcastle Disease (ND) in chickens from an MHC-characterized inbred line. Chickens were experimentally infected with A. galli at 4 weeks of age or left as non-parasitized controls. At 10 and 13 weeks of age half of the chickens were ND-vaccinated and at 16 weeks of age, all chickens were challenged with a lentogenic strain of Newcastle disease virus (NDV). A. galli infection influenced both humoral and cell-mediated immune responses after ND vaccination. Thus, significantly lower NDV serum titres were found in the A. galli-infected group as compared to the non-parasitized group early after vaccination. In addition, the A. galli-infected chickens showed significantly lower frequencies of NDV-specific T cells in peripheral blood three weeks after the first ND vaccination as compared to non-parasitized chickens. Finally, A. galli significantly increased local mRNA expression of IL-4 and IL-13 and significantly decreased TGF-ß4 expression in the jejunum two weeks after infection with A. galli. At the time of vaccination (six and nine weeks after A. galli infection) the local expression in the jejunum of both IFN-? and IL-10 was significantly decreased in A. galli-infected chickens. Upon challenge with the NDV LaSota strain, viral genomes persisted in the oral cavity for a slightly longer period of time in A. galli-infected vaccinees as compared to non-parasitized vaccinees. However, more work is needed in order to determine if vaccine-induced protective immunity is impaired in A. galli-infected chickens.

Chicken mannose-binding lectin (MBL) gene variants with influence on MBL serum concentrations

Mannose-binding lectin (MBL) plays a major role in the innate immune defence by activating the lectin complement pathway or by acting as an opsonin. Two forms of MBL have been characterised from several species, but for humans and chickens, only one form of functional MBL has been described. The human MBL2 gene is highly polymorphic, and it causes varying MBL serum levels. Several of the single-nucleotide polymorphisms (SNPs) have been associated with the severity of diseases of bacterial, viral or parasitic origin. Association between various diseases and different MBL serum levels has also been identified in chickens. In this study, two inbred chicken lines (L10L and L10H) which have been selected for low and high MBL levels in serum and four other experimental chicken lines were analysed for polymorphism in the MBL gene. The presence of polymorphisms in the MBL gene was revealed by southern blot analyses, and the differences in the serum concentrations of MBL were found to be of transcriptional origin according to real-time quantitative reverse transcription PCR analysis. Several SNPs were discovered in the promoter and the 5′ untranslated region of the chicken MBL gene which resulted in the identification of six different alleles. Mapping of regulatory elements in the promoter region was performed, and SNPs that could affect the MBL serum concentration were identified. One SNP that was found to be located in a TATA box was altered in one of the six alleles only. This allele was associated with low MBL serum concentration.

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

Abstract 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.

Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution.

Carboxyfluorescein succinimidyl ester (CFSE) dilution is a well established method for analysis of dividing cells by flow cytometry. In other species the method has been extensively used in the study of antigen-specific T cells. The purpose of this study was to apply the method to chicken peripheral mononuclear blood cells (PBMC) and to evaluate and optimize its performance in relation to detection of vaccine-induced chicken T cells specific for Newcastle disease virus (NDV). The method was based on analysis of CFSE dilution upon ex vivo recall stimulation with whole vaccine antigen. Analysis of proliferation was combined with the use of monoclonal antibodies directed against the lymphocyte surface markers CD4 and CD8 in order to phenotype the responding cells. Problems with nonspecific background proliferation especially in the CD8 compartment were significantly reduced by replacing medium containing fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation lead to a proliferative response in T cells expressing αβ-type TCRs but also those expressing the γδ-type. In summary, the method was found challenging but nevertheless useful to quantify the proliferative response of chicken antigen-specific T cells. Further investigations though, are needed in order to prove what cell subsets are true antigen-specific responders and what cells are bystander activated. Nevertheless, the method is expected to be a valuable tool to evaluate and quantify vaccine responses to current and new chicken vaccines in the future.

Adjuvant effects of mannose-binding lectin ligands on the immune response to infectious bronchitis vaccine in chickens with high or low serum mannose-binding lectin concentrations.

Abstract Mannose-binding lectin (MBL) plays a major role in the immune response as a soluble pattern-recognition receptor. MBL deficiency and susceptibility to different types of infections have been subject to extensive studies over the last decades. In humans and chickens, several studies have shown that MBL participates in the protection of hosts against virus infections. Infectious bronchitis (IB) is a highly contagious disease of economic importance in the poultry industry caused by the coronavirus infectious bronchitis virus (IBV). MBL has earlier been described to play a potential role in the pathogenesis of IBV infection and the production of IBV-specific antibodies, which may be exploited in optimising IBV vaccine strategies. The present study shows that MBL has the capability to bind to IBV in vitro. Chickens from two inbred lines (L10H and L10L) selected for high or low MBL serum concentrations, respectively, were vaccinated against IBV with or without the addition of the MBL ligands mannan, chitosan and fructooligosaccharide (FOS). The addition of MBL ligands to the IBV vaccine, especially FOS, enhanced the production of IBV-specific IgG antibody production in L10H chickens, but not L10L chickens after the second vaccination. The addition of FOS to the vaccine also increased the number of circulating CD4+ cells in L10H chickens compared to L10L chickens. The L10H chickens as well as the L10L chickens also showed an increased number of CD4−CD8α−γδ T-cells when an MBL ligand was added to the vaccine, most pronouncedly after the first vaccination. As MBL ligands co-administered with IBV vaccine induced differences between the two chicken lines, these results indirectly suggest that MBL is involved in the immune response to IBV vaccination. Furthermore, the higher antibody response in L10H chickens receiving vaccine and FOS makes FOS a potential adjuvant candidate in an IBV vaccine.

MHC expression on spleen lymphocyte subsets in genetically resistant and susceptible chickens infected with Marek's disease virus.

Resistance and susceptibility to Mareks disease (MD) are strongly influenced by the chicken major histocompatibility complex (MHC). In this study, splenic lymphocytes from MD-resistant and MD-susceptible chickens of three MHC genotypes (B21/B21, B19/B21, and B19/B19) were analyzed by flow cytometry for MHC surface expression. In the spleen, constitutive MHC class I surface expression was found to be highest in homozygous B19, lowest in homozygous B21, and intermediate in heterozygous B19/B21 animals. This was observed on CD4(+), CD8(+), and Bu-1(+) splenic lymphocytes. Chickens of all three genotypes were subjected to infection with MD virus (GA strain) and spleen samples from infected as well as MHC-matched negative controls were analyzed at 1, 4, and 8 wk post-infection (p.i.). It was observed that MDV induced an increase in MHC class I expression late in the infection. Thus, MHC class I was increased on the surface of CD4(+) cells from infected chickens of all genotypes at 4 and 8 wk p.i. compared with negative controls. Also, MHC class I expression was increased on CD8(+) cells from infected chickens of all genotypes at 4 and 8 wk p.i., except for the homozygous B19 animals, that showed no increase at 8 wk p.i. MDV-induced differences in MHC class II surface levels were also found. Thus, MHC class II expression was increased on CD4(+) cells from infected B19/21 and B21/B21 chickens at 4 wk p.i., and also at 8 wk p.i. on CD4(+) cells from infected B19/B21 animals. MHC class II expression was increased on CD8(+) cells from infected chickens of all genotypes at 4 wk p.i. These findings suggest that MDV infection in vivo increases the level of MHC surface expression on splenic T cells, indicating a possible role in immunity against MDV.