Development of enzyme-linked immunosorbent assay for bovine surfactant protein D in bronchoalveolar lavage fluid.

Surfactant protein D (SP-D) is a pattern recognition molecule that has an important role in pulmonary host defense. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for bovine SP-D and determined the concentration of SP-D in bronchoalveolar lavage fluid (BALF) from calves. Bovine SP-D was purified from BALF using a mannose-Shepharose 6B column. The obtained 44 kDa protein was identified as bovine SP-D by N-terminal amino acid sequence analysis and SDS-PAGE analysis. The peptides corresponding to bovine SP-D amino acid residues SDTRKEGT, which have little homology across bovine serum collectins, were synthesized and used to raise an antibody in rabbits. The obtained antibody was specific for bovine SP-D and did not react with collectins in serum. The anti-bovine SP-D antibody was purified and an ELISA system was developed. The detection range of this assay was 4-125 ng/ml, and the intra-assay and inter-assay coefficients of variation were 5.6 and 9.7%, respectively. The concentrations of SP-D in BALF collected from calves experimentally infected with bovine adenovirus type-3 or Mannheimia haemolytica were determined by the ELISA. Elevation of SP-D was found in BALF from inoculated lobes of infected calves compared with those of non-inoculated lobes and those from control animals. These data suggest that the ELISA developed in this study may be available to investigate the physiological role of bovine SP-D in bovine lung.

Secondary lymphoid areas in calf ileal Peyer's patch.

The secondary lymphoid tissues appear in sheep ileum after involution of ileal Peyer's patch (PP). However, the existence of the secondary lymphoid tissues before involution of ileal PP has not yet been studied. We examined morphological characteristics of the full length of calf ileal PP using gross and microscopic anatomical techniques. Most areas of ileal PP consisted of densely packed lymphatic follicles contained very few follicular T-cell and associated with only scant interfollicular areas. However, the proximal end of ileal PP consisted of widely dispersed lymphatic follicles contained many follicular T-cell and associated with large interfollicular areas. The histological architectures of the proximal end of ileal PP strongly resembled those of the secondary lymphoid tissue in calf.

Histological studies on the ontogeny of bovine palatine and pharyngeal tonsil: germinal center formation, IgG, and IgA mRNA expression.

The development and distribution of lymphocyte subsets in calf palatine and pharyngeal tonsil were examined. During prenatal development, B cells were distributed in the subepithelial area, and T cells and MHC class II(+) cells were found in the deep layer of B-cell area, respectively, in both tonsils. At neonatal stage, lymphoid follicle containing a few CD4(+) cells have been formed in both tonsils. IgG(+) and IgA(+) cells were found in the parafollicular and epithelial area. At 3 months old, many germinal centers were recognized in both tonsils. CD4(+) cells and IgG mRNA expression were detected in light zone of germinal centers. Many IgG, and IgA mRNA expressions also could be detected in the parafollicular and subepithelial area of both tonsils. The data suggest that both tonsils have an important role of local immune defense against invading antigen after birth. The comparison of the histological characteristics of tonsil and Peyer's patch during ontogeny is also discussed.