Effects of encapsulated butyrate and salinomycin on gut leakage and intestinal inflammation in broilers.

1. The objectives of this study were to i) compare the effects of a commercial product providing encapsulated butyrate (EB) in combination with salinomycin in diets of broilers with impaired intestinal integrity and ii) to identify easy-to-measure biomarkers to evaluate intestinal integrity and health.2. In total, 672 one-day-old male broilers (Ross 308) were randomly assigned to three experimental groups (eight replicates/group): no dietary supplement (control); EB (500 mg/kg, UltraGuard™-DUO, Devenish, Ireland); salinomycin (69 mg/kg feed, Sacox® 120). Impaired gut integrity was induced by a 10 times overdose of a commercially attenuated live vaccine against coccidiosis (Hipracox®, Hipra) on d 17 combined with a grower feed providing rye (50 g/kg diet).3. Improved intestinal integrity and functionality were reflected by reduced fluorescein isothiocyanate-dextran (FITC-D) plasma levels, reduced bacterial translocation to the liver (on d 21) and increased plasma colouration level on d 21 after dietary supplementation of salinomycin, compared to a non-supplemented control diet. Both EB and salinomycin reduced plasma levels of D-lactate (P < 0.05).4. An anti-inflammatory effect of salinomycin was indicated as the transient increase in circulating monocytes observed in the EB and control group from 20 to 28 d of age was slightly but not significantly reduced, in the salinomycin-fed group. Interestingly, greater expression of tumour necrosis factor α (TNF-α) and mucin 2 (MUC2) genes (P = 0.039 and P = 0.067, respectively) were detected in the group receiving salinomycin.5. These effects may have collectively contributed to the significantly improved performance of broilers supplemented with salinomycin. The results indicated that EB at 500 mg/kg in feed, in contrast to salinomycin, neither supported gut health nor modulated intestinal integrity in broilers.

Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease.

The aim of this study was to optimise and evaluate an intracellular cytokine staining (ICS) assay for assessment of T cell IFN-γ responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-γ using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-γ production in chicken peripheral blood leucocytes. Chickens from an inbred white leghorn line containing two MHC haplotypes, B19 and B21, were divided into three experimental groups; one group was kept as naive controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3+TCRγδ+ (γδ T cells) cells and CD3+TCRγδ- cells (αß T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing cells were CD3- indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3+TCR1γδ-CD8α+ (CTL) population produced the highest NDV specific IFN-γ responses, with significantly elevated levels of IFN-γ producing cells in the B19 chickens vaccinated orally with live attenuated NDV vaccine. This was not the case in the B21 animals, indicating a haplotype restricted variation. In contrast, the CD3+TCR1γδ-CD4+ (Th) population did not show a significant increase in IFN-γ production in NDV stimulated samples which was in part due to a high number of IFN-γ producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-γ producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation.

Comparison of growth performance and immune parameters of three commercial chicken lines used in organic production.

Owing to the higher demands for avoiding medication and antibiotics, health status of the production animals plays an important role in the poultry industry, especially in organic poultry systems. Immunity plays a major role in keeping the host free from disease, and it is evident that the host's genetic make-up influences immunity and disease resistance/susceptibility in chickens. Previously, breeding strategies aimed at selection for resistance against specific diseases with the risk of creating less disease resistance against other pathogens. Changing breeding strategies towards selection of chickens with a more general and broad disease resistance or robustness may therefore improve the overall health status, animal welfare, and food security in the poultry production. The aim of this study was therefore to compare the immunocompetence of the presumed "robust" Hellevad chickens with two chicken lines widely used in organic production, Bovans Brown (Bovans) and Hisex White (Hisex). The chickens were subjected to a routine vaccination program comprising one parasite and four viral vaccines. The current study indicates that considerable differences in immunocompetence may exist between commercial layer lines used in organic production. The Hellevad chickens were found to have higher body weight at the end of the experiment (17 weeks of age) than the other two lines. Furthermore, Hellevad and Hisex chickens were found to have higher levels of humoral innate immunity with regard to sample to positive ratio of natural antibodies in serum and concentration of mannose-binding lectin in serum as compared to Bovans. Moreover, indications of an inflammatory response were observed in the Bovans at week 5, corresponding to 1 week after vaccination with live infectious bursal disease virus. With regard to adaptive immune parameters such as IgY concentration in blood and infectious bursal disease virus (IBDV)-specific antibody titres, the Hellevad and Hisex chickens had lower levels than the Bovans. How the differences observed in growth and immune parameters in the three chicken lines influence the immune protection against infection needs to be studied further.

Monitoring of local CD8ß-expressing cell populations during Eimeria tenella infection of naïve and immune chickens.

The purpose of this study was to monitor abundance and activation of local CD8ß-expressing T-cell populations during Eimeria tenella infections of naïve chickens and chickens immune by previous infections. Chickens were infected with E. tenella up to three times. Caecal T-cell receptor (TCR) γ/δ-CD8ß+ cells (cytotoxic T lymphocytes; CTL) and TCRγ/δ+CD8ß+ cells were characterized with respect to activation markers (blast transformation, CD25 and cell surface CD107a). Cells were also induced to degranulate in vitro as a measure of activation potential. Major findings included a prominent long-lasting, up to 6 weeks, increase in the proportion of CTL among caecal CD45+ cells in the later stages after primary E. tenella infection. These CTL also showed clear signs of activation, that is blast transformation and increased in vitro induced degranulation. At second and third E. tenella infection, chickens showed strong protective immunity but discrete signs of cellular activation were observed, for example increased in vitro induced degranulation of CTL. Thus, primary E. tenella infection induced clear recruitment and activation of local CTL. Upon subsequent infections of strongly immune chickens cellular changes were less prominent, possibly due to lower overall numbers of cells being activated because of the severe restriction of parasite replication.

Transcription efficiency of different chicken mannose-binding lectin promoter alleles.

The serum collectin mannose-binding lectin (MBL) plays a major role in innate immunity by activation of the lectin complement pathway or by acting as an opsonin. The serum levels of human and animal MBL are associated with susceptibility to a wide range of infections, and the variation of MBL in serum is genetically determined. In the chicken, 14 single nucleotide polymorphisms (SNPs) have so far been found in the MBL promoter region. In this study, the transcription activity of a 670-bp promoter region covering all 14 SNPs from the four MBL promoter alleles A1 to A4 was assessed using a dual-luciferase assay. Of the analysed alleles, A1 showed the highest transcription activity although this allele is frequently found in chickens with low MBL mRNA expression.

Acquisition of resistance after continuous infection with Ascaridia galli in chickens.

SUMMARY Acquired resistance against Ascaridia galli infection was studied in seventy-two 18-week-old white Leghorn chickens allocated to six groups (G1-G6). In order to understand the population dynamics following trickle-infection (100 eggs per chicken twice weekly), chickens of subgroups of G1 were necropsied 3 days after 1, 6 or 12 inoculations (G1A, G1B and G1C respectively), while G2-G4 were inoculated for 6 weeks. G2 was necropsied 4 weeks after the last inoculation. The number of established larvae increased initially (between G1A and G1B) but decreased after repeated inoculations (G1C, G2). G3, G4 and G5 were used to measure the efficacy of anthelminthic treatment and to monitor the acquisition of resistance following a challenge infection. At week 7 G3, G4 and G5 were treated with flubendazole for 7 days in the feed. Two weeks after treatment the chickens in G4 and G5 were challenged with 500 eggs. G6 was left as uninfected control. Necropsy at week 10 after first inoculation revealed a lower establishment rate, an impaired development and a more posterior localization of the larvae in G4 (trickle-infected-treated-challenged) compared with G5 (treated-challenged). IgY level in serum reached noticeable level at 14 dpi in G2 and G4 chickens, and in G4 chickens IgY level further increased after challenge infection. The study provides evidence that acquired resistance against A. galli in chickens leads to a significant yet incomplete protection against re-infection.

The effects of feed restriction on physical activity, body weight, physiology, haematology and immunology in female mink.

The aim of the present study was to investigate if adult mink females characterised as having a high or low residual feed intake (RFI) differed in their response to feed restriction with regard to activity, body weight loss and physiological parameters. For RFI-High, the activity was higher prior to the expected feeding time both in the cases of restrictive and ad libitum feeding indicating a changed RFI-High feeding motivation and a higher risk of developing stereotypic activity. The body weight gain and the feed consumption were higher for RFI-High than for RFI-Low when feeding ad libitum indicating that RFI-High has a higher growth potential and/or a higher energy requirement than RFI-Low. Signs of immunosuppression were shown in connection with restrictive feeding compared to ad libitum feeding, and RFI-High females seemed to be more susceptible to immunosuppression than RFI-Low females. Based on the present results, mink characterised as RFI-Low would be preferable as breeders because they involve lower feed costs and seem to be less susceptible to immunosuppression. They also seem to have less risk of developing stereotypic activity, which is beneficial for the welfare.

Assessment of Newcastle disease-specific T cell proliferation in different inbred MHC chicken lines.

In this study, we have described the establishment of an antigen-specific T cell proliferation assay based on recall stimulation with Newcastle disease (ND) antigen; further, we have described the results obtained after recall stimulation of animals containing different major histocompatibility complex (MHC) haplotypes, vaccinated against ND. First optimization of the assay was performed to lower unspecific proliferation and to enhance antigen-specific T cell proliferation. These two issues were achieved using ethylene diamine tetra acetic acid as stabilizing agent in blood samples and autologous immune serum in culture medium. The optimized assay was used to screen chickens with different MHC haplotypes for their ability to perform T cell proliferation. Results showed that the antigen-specific response of CD4(+) and CD8(+) T cells from B12 chickens was generally low, whereas B13, B130 and B201 chickens were medium in CD4(+) or CD8(+) T cell responses. High responses were seen only in few animals of each haplotype and not in general. A polymorphism in the chicken CD8α gene was found in our experimental chicken lines, resulting in incapability to detect CD8α(+) T cells using antibodies from the CT8 clone. Screening chickens with alternative antibodies showed that antibodies from the 2-398 clone were able to discriminate all CD8α(+) cells from CD8α(-) cells, and consequently this antibody was used in a second vaccination experiment performed with chickens of the haplotypes B13 and B130. This experiment showed a significant difference in antigen-specific proliferation of CD4(+) T cells between the two lines, but not in CD8α(+) T cell proliferation.

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 αß-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.

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.

Immune response to a killed infectious bursal disease virus vaccine in inbred chicken lines with different major histocompatibility complex haplotypes.

The influence of MHC on antibody responses to killed infectious bursal disease virus (IBDV) vaccine was investigated in several MHC inbred chicken lines. We found a notable MHC haplotype effect on the specific antibody response against IBDV as measured by ELISA. Some MHC haplotypes were high responders (B201, B4, and BR5), whereas other MHC haplotypes were low responders (B19, B12 and BW3). The humoral response of 1 pair of recombinants isolated from a Red Jungle Fowl (BW3 and BW4) being identical on BF and BG, but different on BL, indicated that part of the primary vaccine response was an MHC II restricted T-cell dependent response. The humoral response in another pair of recombinant haplotypes originating in 2 different White Leghorn chickens being BF21, BL21, BG15 (BR4) and BF15, BL15, BG21 (BR5) on the MHC locus indicated that the BG locus may perform an adjuvant effect on the antibody response as well. Vaccination of chickens at different ages and in lines with different origin indicated that age and background genes also influence the specific antibody response against inactivated IBDV vaccine.

Molecular characterization of major histocompatibility complex class I (B-F) mRNA variants from chickens differing in resistance to Marek's disease.

In this study, the relative distributions of two alternatively polyadenylated chicken major histocompatibility complex (MHC) mRNA isoforms of approximately 1.5 and 1.9 kb were analysed in spleen cells from chickens homozygous for the MHC haplotypes B21 and B19v1 as well as in heterozygous B19v1/B21 birds. Both isoforms are likely to encode classical MHC class I (B-F) alpha chains. The B19v1 and B21 MHC haplotypes confer different levels of protection against Marek's disease (MD), which is caused by infection with MD virus (MDV). In spleen cells, MD-resistant B21 birds were shown to have the highest percentage of the 1.5 kb variant relative to the total MHC class I expression, MD-susceptible B19v1 birds the lowest and B19v1/B21 birds an intermediate percentage. Infection of 4-week-old chickens with the GA strain of MDV was shown to cause a significant increase in the relative amount of 1.5 kb transcripts in B21 birds 32 days postinfection (dpi). Alternatively polyadenylated mRNA isoforms may encode identical proteins, but differences in the 3' untranslated region (UTR) can influence polyadenylation, mRNA stability, intracellular localization and translation efficiency. It was shown that the increased 1.5 kb percentage in B21 birds 32 days postinfection may be a result of a change in the choice of poly(A) site rather than a locus-specific upregulated transcription of the BF1 gene that preferentially expresses the 1.5 kb variant. Furthermore, the 3' end of the 1.5 kb mRNA variants deriving from B19v1 and B21 chickens was characterized by Rapid Amplification of cDNA Ends (RACE) and sequencing. No potentially functional elements were identified in the 3' UTR of the RACE products corresponding to this short isoform. However, variation in polyadenylation site was observed between the BF1 and BF2 mRNA transcripts and alternative splicing-out of the sequence (exon 7) encoding the second segment of the cytoplasmic part of the mature BF2*19 molecules. This alternative exon 7 splice variant was also detected in other MD-susceptible haplotypes, but not in the MD-resistant B21 and B21-like haplotypes, suggesting a potential role of exon 7 in MHC-related MD resistance.

Differences in chicken major histocompatibility complex (MHC) class Ialpha gene expression between Marek's disease-resistant and -susceptible MHC haplotypes.

The expression of chicken major histocompatibility complex (MHC) class Ialpha genes was investigated in spleen cells from a panel of chickens with well-defined MHC haplotypes, and two class Ialpha transcripts of 1.9 and 1.5 kb were detected in various amounts. In BW1, B130 and B21, the two transcripts were almost equally expressed. In B2, B6, B12 and B19, the ratio between the two transcripts was 4 : 1, with the 1.9 kb transcript having the strongest expression. In B14 and B15, the 1.5 kb transcript was undetectable and the 1.9 kb transcript appeared to be exclusively expressed. Thus, haplotypes considered to have an MHC-determined resistance to Marek's disease (MD) had the highest relative amount of the 1.5 kb transcript, whereas haplotypes considered to be MD-susceptible had the lowest. In order to address a possible correlation between MHC-Ialpha transcriptional patterns and MD resistance, a larger animal material experimentally infected with MD virus (MDV) was examined. The expression of MHC class Ialpha genes was investigated in spleens as well as in other organs, 9 weeks post-infection (p.i.), from animals of the two MD-resistant haplotypes B21 and BW1 as well as from the MD-susceptible haplotype B19. In the spleen cells of infected animals, the relative amount of the 1.5 kb transcript in the haplotypes BW1 and B21 was shown to be significantly higher than that in B19. Interestingly, in infected BW1 and B21 animals, the relative amount of the 1.5 kb transcript was also significantly higher than that in healthy MHC-matched controls. In B19, no differences were detected between uninfected and infected animals. Furthermore, it was shown in BW1 and B21 that the two classical MHC-Ialpha genes located in the MHC region were both able to produce both mRNA transcripts. Hybridization experiments, using specific probes upstream and downstream of the polyadenylation signals in the 3' end of the MHC-Ialpha genes, demonstrated that alternate use of these signals is probably involved in the production of the two mRNA transcripts.

Major histocompatibility complex-linked immune response of young chickens vaccinated with an attenuated live infectious bursal disease virus vaccine followed by an infection.

The influence of the MHC on infectious bursal disease virus (IBDV) vaccine response in chickens was investigated in three different chicken lines containing four different MHC haplotypes. Two MHC haplotypes were present in all three lines with one haplotype (B19) shared between the lines. Line 1 further contains the BW1 haplotype isolated from a Red Jungle Fowl. Line 131 further contains the B131 haplotype isolated from a meat-type chicken. Finally, Line 21 further contains the international B21 haplotype. The chickens were vaccinated with live attenuated commercial IBDV vaccine at 3 wk of age, followed by a challenge with virulent IBDV at 6 wk of age. In this study, we found a notable MHC haplotype effect on the specific antibody response against IBDV, as measured by ELISA. The BW1 haplotype was found to have a significantly higher serum antibody titer against IBDV (7,872) than haplotypes B19 (mean 5,243), B21 (5,570), and B131 (5,333) at 8 d postinfection. However, a virus-neutralizing antibody test did not reflect this result. Nevertheless, the MHC haplotype-associated protective immunity was further supported by the bursa of Fabricius (bursa) recovery from the disease, as measured by histological scorings of the bursa. Chickens carrying the BW1 haplotype had a significantly lower bursa lesion score (1.7) than the haplotypes B19 (mean 3.8), B21 (3.6), and B131 (4.3) 8 d postinfection. Furthermore, multiple line effects were found in other variables when comparing Day 6 with Day 8. Body weight, relative weights of the bursa and the spleen, percentage and relative number of MHC II molecules on MHC II-positive lymphocytes, percentage and relative number of CD4 molecules on CD4-positive lymphocytes, and the specific antibody response all differed significantly among lines. Line 1, with Red Jungle Fowl genes, was clearly differentiated from the other two investigated lines. These results suggest an MHC II restricted T-cell dependent secondary antibody response against IBDV.

Molecular characterization of major and minor MHC class I and II genes in B21-like haplotypes in chickens.

The major histocompatibility complex (MHC) sequences of three B21-like haplotypes deriving from very different origins including the Red Jungle Fowl Gallus Gallus gallus were compared with the MHC sequences of the standard B21 haplotype from Scandinavian White Leghorn Gallus domesticus. The present analysis reveals two cDNA sequences for B-F and two cDNA sequences for B-LB for every B21-like haplotype, including B21 itself. Contrary to expectation, no sequence polymorphism in the antigen-binding domains of the MHC genes, between the investigated haplotypes, was found. The relative level of MHC class I molecules on the surface of leukocytes measured by flow cytometry was also analysed and found to be low in Marek's Disease (MD)-resistant B haplotypes (B21 and B21-like) and high in MD-susceptible B haplotypes (B15 and B19). However, in heterozygous (resistant/susceptible) animals, the relative level was almost as high as in susceptible haplotypes.

A polymorphic major histocompatibility complex class II-like locus maps outside of both the chicken B-system and Rfp-Y-system.

Chickens have two major regions encoding major histocompatibility complex (MHC) class Ialpha genes and MHC class IIss genes, the serological and functional B-system and the Rfp-Y-system. Recently, they have been shown to assort in a genetically independent way although still located on the same microchromosome. Moreover, the monomorphic MHC class IIalpha gene maps at a third locus located 5 cM from the nearest class IIss genes, located in the B-system (Kaufman et al., 1995). A pedigree family was studied in three generations in order to assign MHC class IIss restriction fragments observed in Southern blot analyses to either the B-system, the Rfp-Y-system or the B-Lalpha locus. In this study, we demonstrate by classical genetic testing of chickens within this fully pedigreed family the existence of an MHC class II-like polymorphic restriction fragment that segregates independently of the B-system, the Rfp-Y-system and of the B-Lalpha locus.

Cloning and sequencing of a cDNA encoding chicken mannan-binding lectin (MBL) and comparison with mammalian analogues.

The serum lectin, mannan-binding lectin (MBL) (also denoted mannan-binding protein or mannose-binding protein, MBP) has been identified in mammals (humans, monkey, cow, rabbit, mouse and rat). Upon binding to carbohydrates on the surface of microorganisms, MBL mediates activation of the complement system, leading to killing of the microorganism. MBL thus exerts a role in the innate immune defence. We have described the isolation and partial characterization of an analogous protein in chicken serum. Oligonucleotides based on the N-terminal sequence of this protein were used in a reverse transcription-polymerase chain reaction (RT-PCR) with chicken liver RNA as template. The PCR product was sequenced and found to encode part of the NH2 terminus of chicken MBL. A perfect match probe was synthesized and used to screen a chicken liver cDNA library. The isolated clones carried a cDNA insert of 1692 bp with an open reading frame of 714 bp encoding a mature protein of 238 amino acids including a signal peptide of five amino acids. The deduced amino acid sequence agrees with those determined by conventional amino acid sequence analysis of the peptides except for four residues. We have compared the deduced primary structure of chicken MBL with the mammalian analogues. The phylogenetic analysis indicates that the gene duplication leading to two different MBL forms in mammals occurred after the split from birds and reptiles. This concurs with the finding of only one form of MBL in chickens.