Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines.

Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is a particular interest in developing robust high-throughput assays as chicken vaccine trials usually comprise many individuals. In many respects, the avian immune system differs from the mammalian, and T cell assessment protocols must be adjusted accordingly to account for, e.g., differences in leukocyte subsets.The carboxyfluorescein succinimidyl ester (CFSE) method described in this chapter has been adapted to chicken cells. In this test, cells of interest are stained with CFSE. The succinimidyl ester group covalently binds to cellular amines forming fluorescent conjugates that are retained in the cells even throughout division. This leads to daughter cells containing half the fluorescence of their parents. When lymphocytes are loaded with CFSE prior to ex vivo stimulation with specific antigen, the measurement of serial halving of its fluorescence by flow cytometry identifies the cells responding to the stimulation. This method has been successfully applied to studies of chicken antigen-specific T cells.

RNA sequencing-based analysis of the spleen transcriptome following infectious bronchitis virus infection of chickens selected for different mannose-binding lectin serum concentrations.

BACKGROUND: Avian infectious bronchitis is a highly contagious disease of the upper-respiratory tract caused by infectious bronchitis virus (IBV). Understanding the molecular mechanisms involved in the interaction between innate and adaptive immune responses to IBV infection is a crucial element for further improvements in strategies to control IB. To this end, two chicken lines, selected for high (L10H line) and low (L10L line) serum concentration of mannose-binding lectin (MBL) were studied. In total, 32 birds from each line were used. Sixteen birds from each line were infected with IBV and sixteen were left uninfected. Eight uninfected and infected birds from each line were euthanized at 1 and 3 weeks post infection. RNA sequencing was performed on spleen samples from all 64 birds and differential gene expression analysis was performed for four comparisons: L10L line versus L10H line for uninfected birds at weeks 1 and 3, respectively, and in the same way for infected birds. Functional analysis was performed using Gene Ontology (GO) Immune System Process terms specific for Gallus gallus. RESULTS: Comparing uninfected L10H and L10L birds, we identified 1698 and 1424 differentially expressed (DE) genes at weeks 1 and 3, respectively. For the IBV-infected birds, 1934 and 866 DE genes were identified between the two lines at weeks 1 and 3, respectively. The two most enriched GO terms emerging from the comparison of uninfected birds between the two lines were "Lymphocyte activation involved in immune response" and "Somatic recombination of immunoglobulin genes involved in immune response" at weeks 1 and 3, respectively. When comparing IBV-infected birds between the two lines, the most enriched GO terms were "Alpha-beta T cell activation" and "Positive regulation of leukocyte activation" at weeks 1 and 3, respectively. CONCLUSIONS: Healthy birds from the two lines showed significant differences in expression profiles for subsets of adaptive and innate immunity-related genes, whereas comparison of the IBV-infected birds from the two lines showed differences in expression of immunity-related genes involved in T cell activation and proliferation. The observed transcriptome differences between the two lines indicate that selection for MBL had influenced innate as well as adaptive immunity.

Annotation and genetic diversity of the chicken collagenous lectins.

Collectins and ficolins are multimeric proteins present in various tissues and are actively involved in innate immune responses. In chickens, six different collagenous lectins have been characterized so far: mannose-binding lectin (MBL), surfactant protein A (SP-A), collectin 10 (COLEC10), collectin 11 (COLEC11), collectin 12 (COLEC12), lung lectin (LL) and one ficolin (FCN). However, the structural and functional features of the chicken collectins and ficolin are still not fully understood. Therefore, the aims of this study were: (i) to make an overview of the genetic structure and function of chicken collectins and the ficolin, (ii) to investigate the variation in the chicken collectins and the ficolin gene in different chicken populations, and (iii) to assess the presence of MBL gene variants in different chicken populations. We performed comparative genomic analysis using publically available data. The obtained results showed that collectins and ficolins have conserved protein sequences and gene structure across all vertebrate groups and this is especially notable for COLEC10, COLEC11 and COLEC12. For the purpose of studying the genetic variation, 179 animals from 14 populations were genotyped using 31 SNPs covering five genomic regions. The obtained results revealed low level of heterozygosity in the collagenous lectins except for the COLEC12 gene and the LL-SPA-MBL region compared to heterozygosity at neutral microsatellite markers. In addition, the MBL gene variants were assessed in different chicken populations based on the polymorphisms in the promoter region. We observed 10 previously identified MBL variants with A2/A8 and A4 as the most frequent alleles.

A two-nucleotide deletion renders the mannose-binding lectin 2 (MBL2) gene nonfunctional in Danish Landrace and Duroc pigs.

The mannose-binding lectins (MBLs) are central components of innate immunity, facilitating phagocytosis and inducing the lectin activation pathway of the complement system. Previously, it has been found that certain single-nucleotide polymorphisms (SNPs) in porcine MBL1 and MBL2 (pMBL1, pMBL2) affect mRNA expression, serum concentration, and susceptibility to disease, but the combinatory effect of pMBL1 and pMBL2 genotypes needs further elucidation. In the present study, pMBL1 and pMBL2 alleles, combined pMBL haplotypes, and MBL-A concentration in serum were analyzed in purebred Landrace (N = 30) and Duroc (N = 10) pigs. Furthermore, the combined pMBL haplotypes of 89 Piètrain × (Large White × Landrace) crossbred pigs were studied, and the genotypes of 67 crossbreds challenged with Escherichia coli were compared to their individual disease records. In the purebred animals, three non-synonymous SNPs and a two-nucleotide deletion were detected in the coding sequence of pMBL2. The two-nucleotide deletion was present at a frequency of 0.88 in the Landrace pigs and 0.90 in the Duroc pigs, respectively. In the crossbreds, the T allele of the SNP G949T in pMBL1-previously shown to have profound effect on MBL-A concentration even in the heterozygote condition-was detected in 47 % of the animals. Finally, an association was found between low-producing MBL genotypes and low body weight on the day of weaning in the same animals.

Correlation in chicken between the marker LEI0258 alleles and Major Histocompatibility Complex sequences.

BACKGROUND: The LEI0258 marker is located within the B region of the chicken Major Histocompatibility Complex (MHC), and is surprisingly well associated with serology. Therefore, the correlation between the LEI0258 alleles and the MHC class I and the class II alleles at the level of sequences is worth investigating in chickens. Here we describe to which extent the LEI0258 alleles are associated with alleles of classical class I genes and non-classical class II genes, in reference animals as well as local breeds with unknown MHC haplotypes. METHODS: For the class I region, in an exploratory project, we studied 10 animals from 3 breeds: Rhode Island Red, White Leghorn and Fayoumi chickens, by cloning and sequencing B-F1 and B-F2 cDNA from exon 1 to 3'UTR. For the class II region, we reconstructed haplotypes of the 8.8 kb genomic region encompassing three non-classical class II genes: B-DMA, B-DMB1 and B-DMB2, for 146 animals from more than 50 breeds including wild species of jungle fowls. RESULTS: Overall we found that the LEI0258 marker genotypes gave good indications of the MHC haplotypes, and a very good predictions (>0.95) of the heterozygosity of an animal at the MHC locus. CONCLUSIONS: Our results show that the LEI0258 alleles are strongly associated with haplotypes of classical class I genes and non-classical class II genes, unravelling the reasons why this marker is becoming the reference marker for MHC genotyping in chickens.

Colostrum and bioactive, colostral peptides differentially modulate the innate immune response of intestinal epithelial cells.

Characterization and identification of peptides with bioactivity from food have received considerable interest recently since such bioactive components must be adequately documented if they are part of functional food claims. We have characterized peptides from colostrum or those generated by a simulated gastrointestinal digest (GI) and tested them for bioactivity using murine intestinal (mIC(c12)) cells and compared with bioactivity of intact colostrum. The peptides were recovered in the permeate after dialysis. The presence of peptides in the permeate was confirmed by C(18) RP-HPLC, determination of free amino termini and MALDI MS. The bioactivity of the intact colostrum and colostral peptides in the permeate was tested using mIC(c12) cells stimulated in the absence or presence of different bacterial ligands that mediate cellular activation through stimulation of Toll-like receptors (TLR). Whereas intact colostrum generally reduced TLR-mediated signaling, the isolated peptides seemed to either stimulate or reduce the immune response depending on the bacterial ligand used for stimulation. Interestingly, the most potent bioactive peptides originated from nondigested colostrum, which had only been subject to endogenous protease activity. Identified peptides in the nondigested colostrum originated exclusively from the casein fraction of colostrum as shown by MALDI MS/MS identification. Thus, multiple components with different bioactivities towards the innate immune response appear in bovine colostrum.

A novel, simple and sensitive ligand affinity capture method for detecting molecular interactions by MALDI mass spectrometry.

A simple and sensitive ligand affinity capture method (LAC) was developed to detect biotinylated biomolecules bound to a biotin-avidin base by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI ToF MS). Glass slides covered with a metal film for MALDI MS applications were treated with amino-silane and derivatized with biotin followed by binding of avidin. Washing buffers with high ionic strength increased the specificity of the subsequent binding of biotinylated biomolecules to the avidin layer. A combined thin layer-dried droplet method using alpha-cyano-4-hydroxycinnamic acid (CHCA) in acetone or ethyl acetate resulted in the most intense ions of biotinylated polymyxin B, whereas the matrix conditions did not influence the detection of angiotensin II. Addition of biotinylated biomolecules in the low femtomole to low picomole range resulted in sufficient ion intensity for detection by the LAC method. The LAC concept was extended by binding of biotinylated lipopolysaccharide to the biotin-avidin base followed by preferential capture and specific detection of the binding antagonist polymyxin B.