Fig. 1. Flow cytometric analysis of bovine peripheral blood neutrophils stained with FITC-M haemolytica adhesin. (A) Bovine neutrophils: Dotted line indicates the background level without MhA-FITC; dark line indicates the fluorescence level of the MhA positive cells. (B) Bovine neutrophils treated previously with C. perfringens sialidase that showed identical fluorescence intensity with MhA positive cells (dark line) and dotted line indicates control without MhA. Thin line, in panels A and B, indicates the cells treated with MhA incubated previously in 200 mM GlcNAc. Representative results from one experiment out of five.

Fig. 2. Purification of the receptor for M. haemolytica adhesin from bovine neutrophils. Neutrophils lysate was purified on MhA-Sepharose 4B column. The unretained fraction was eluted with PBS-T (PBS-0.1% TritonX-100) and the affinity purified receptor was eluted by addition of 200 mM GlcNAc in PBS-T. Optical density at 280 nm was determined on fractions dialyzed against PBS.

Fig. 3. SDS-PAGE and Western blot of the purified MhA-neutrophils receptor. Lane A, molecular weight markers. Lane B, 50 μg of bovine leukocytes (1 × 10⁷) cell lysate. Lane C, 50 μg of neutrophils lysate not retained by the MhA-Sepharose column. Lane D, 10 μg of purified MhA-neutrophils receptor. Lane E, Western blot analysis of neutrophils lysate separated on SDS-PAGE and transferred to nitrocellulose. The nitrocellulose membranes were incubated with peroxidase-MhA (10 μg/mL) and revealed with O-phenylene-diamine and H₂O₂ in 100 mM citrate buffer, pH 5.6. Lane F, peroxidase-MhA (10 μg) was incubated with 200 mM GlcNAc before being added to the transferred neutrophils lysate. The molecular weight markers are: myosin (200 kDa); β-galactosidase Escherichia coli (116 kDa); phosphorylase B (97 kDa); bovine serum albumin (66 kDa); ovalbumin (45 kDa); carbonic anhydrase (31 kDa); trypsin inhibitor (21 kDa).

Amino acid composition of the receptor for the adhesin from Mannheimia haemolytica from bovine neutrophils

Carbohydrate composition of the receptor for the adhesin from Mannheimia haemolytica from bovine neutrophils

Nitroblue tetrazolium (NBT) reduction by bovine peripheral blood neutrophils (4 × 10⁵ cells)

Numerical values represent the mean ± SD of five experiments.Significant difference (p < 0.005) determined by Mann–Whitney U test observed when the effect was compared with NBT treated cells and (p < 0.05) when compared with the effect induced with MhA. GlcNAc at 200 mM. The MhAr was desialylated with C. perfringes sialidase, and deglycosylated forms of the receptor were obtained after almond PNGase (PNGase MhAr) or O-glycanase (O-GlycMhAr) treatments. Phorbol myristate acetate (PMA) and NBT (0.2%)–Zymosan (0.1%) were used as control of positive stimulation. MhAr at 10 μg/mL showed no effect on NBTr produced after Zymosan-NBT or PMA activation.