Serum levels, ontogeny and heritability of chicken mannan-binding lectin (MBL).

Mannan-binding lectin (MBL) is a serum lectin found in mammals and recently also in birds. It is thought to play an important role in the innate immune defence through binding to surface carbohydrates on micro-organisms followed by complement activation via the MBL pathway. This results in opsonization or direct complement-mediated killing. To gain further knowledge about the physiology and function of the protein, we developed an enzyme-linked immunosorbent assay for chicken MBL and used this to investigate the level of MBL in different chicken strains during embryogenesis, early and adult life. The MBL concentrations in 308 chickens, representing 14 different strains, showed a non-Gaussian, unimodal distribution profile with a mean concentration of 5.8 micrograms/ml (range 0.4-37.8 micrograms/ml). No difference between the strains could be demonstrated and no chickens were found deficient in MBL. Ontogenetic studies showed that MBL is already detectable in embryos at a gestational age of 10 days (11 days before hatching). At hatching, the level is comparable to the level found in adult chickens. This level is fairly stable during the first weeks of life, but a deficiency state develops at 4 weeks of age, whereafter the level is normalized again at 5 weeks of age. Chickens with relatively low or high MBL levels were bred with cockerels having similar MBL levels and this resulted in F1 generations with significantly different MBL levels, suggesting that the protein level is genetically influenced.

Immunohistochemical investigation of the tissue distribution of mannan-binding lectin in non-infected and virus-infected chickens.

This paper describes the results of immuno-histochemical staining for chicken mannan-binding lectin (MBL) in formalin-fixed tissue sections from non-infected chickens, and from chickens infected with infectious laryngotracheitis virus (ILTV) or infectious bursal disease virus (IBDV). In the non-infected chickens, MBL was detected in the cytoplasm of a few hepatocytes and in the germinal centres of the caecal tonsils, whereas sections of kidney, heart muscle, spleen, cerebrum, thymus, adrenal gland, bursa of Fabricius, bone marrow and trachea were without staining. In the ILTV-infected chickens, an intense staining reaction for MBL was detected in the cytoplasm of all hepatocytes and on the surface of, and inside, ILTV-infected cells. Also in the IBDV-infected chickens, an intense staining reaction for MBL was detected in the cytoplasm of all hepatocytes. No staining was seen in the follicles of the bursa of Fabricius, but MBL was present in non-identified cells in the interstitium, and in the cytoplasm of macrophage-like cells, located peripheral to the ellipsoid of the spleen. These findings indicate the liver as the primary site of MBL synthesis, and points to up-regulation as a result of the viral infections. The location outside the liver could indicate a role of MBL in the immune defence.

Inflammatory response of different chicken lines and B haplotypes to infection with infectious bursal disease virus.

Chickens representing two different inbred lines (layer and meat-type) and three different B haplotypes (BW1, B19 and B131) were infected with infectious bursal disease virus (IBDV) at 21 days of age. Mortality was recorded, and surviving chickens were killed and examined either 3 or 17 days post-infection. Non-infected control chickens were run in parallel. The variables recorded were all significantly changed by the virus infection, including the serum concentration of mannan-binding lectin that was increased 2-fold 3 days post-infection. A significantly increased mortality, liver to body weight ratio and erythrocyte sedimentation rate (ESR) was seen among chickens from the layer type line compared with the meat-type line. In addition, the haplotype of the chickens influenced the atrophy and hypertrophy of the thymus. It was concluded that the meat-type chicken line was more resistant to IBDV infection than the layer-type line, and that mortality rate, liver to body weight ratio and ESR were valuable variables for evaluation of the level of IBDV infection-induced inflammation and disease.

New chicken Rfp-Y haplotypes on the basis of MHC class II RFLP and MLC analyses.

New chicken Rfp-Y haplotypes were determined by the use of restriction fragment length polymorphism (RFLP) and mixed lymphocyte culture (MLC) in four different chicken haplotypes, B15, B19, B21, B201. The RFLP polymorphism was mapped to the Rfp-Y system by the use of a subclone (18.1) which maps near a polymorphic lectin gene located in the Rfp-Y system and DNA from families with known segregation of the implicated RFLP polymorphism. For the first time it is shown that major histocompatibility complex class II genes in the Rfp-Y system have functional implications. Sequence information of the B1 domain of the proposed Rfp-Y haplotypes was obtained which supported the functional data.

Collectin in a non-mammalian species: isolation and characterization of mannan-binding protein (MBP) from chicken serum.

A chicken serum lectin was isolated by affinity chromatography on TSK-75 beads derivatized with the monosaccharide N-acetyl-D-mannosamine (ManNAc). Serum was applied to the column in a Ca(2+)-containing buffer and proteins were eluted with EDTA. After recalcification, the eluate was passed through a new ManNAc-derivatized column. Bound proteins were eluted with 50 mM ManNAc. Anti-carbohydrate antibodies present in the eluate were removed by passage through a rabbit anti-chicken immunoglobulin derivatized column, and the lectin was further purified by ion-exchange chromatography and gel-permeation chromatography. The purified chicken lectin shows an overall structure similar to mammalian mannan-binding protein (MBP). SDS-PAGE revealed two polypeptides of M(r) 33 and 34 kDa (reduced) with identical sequence for the first 30 NH2-terminal residues. The NH2-terminal sequence shows 43% identity with the human MBP. Like mammalian MBP, the polypeptides of the chicken lectin are degraded by treatment with collagenase. Residues 26-30 (G-L-P(OH)-G-D) are likely to represent the beginning of the collagenous region. Mobilities on SDS-PAGE of the COOH-terminal collagenase-resistant fragment under reduced and non-reduced conditions indicate the presence of intrachain disulphide bonds, as are also found in mammalian MBP. Gel chromatography showed an intact mol. wt of 750 kDa. Binding of the chicken MBP to mannan was inhibited by monosaccharides in the following order of potency: ManNAc > L-fucose > mannose > N-acetylglucosamine. Other monosaccharides inhibited poorly or not at all. Chicken MBP, bound to mannan, activated the classical complement pathway in human serum. Electron micrographs show structures and dimensions resembling human MBP. Overall, the results show that the purified lectin is the chicken homologue to mammalian MBP and indicate the presence of a MBP-like clearance system outside mammals.

Restriction fragment length polymorphism analysis of the chicken B-F and B-L genes and their association with serologically defined B haplotypes.

Seven serologically defined chicken haplotypes have been analysed by restriction fragment length polymorphism (RFLP) with chicken cDNA probes specific for MHC class I and II. The results demonstrate an excellent correlation between the observed RFLP banding patterns in the investigated haplotypes and the serological B-typing. In future, RFLP analysis in addition to serological B-typing may sharpen the tools in the search for recombinant chromosomes separating B-F and B-L.