Comparison between Mycoplasma bovoculi strains by electrophoretic and immunoblotting techniques: identification of membrane proteins involved in adherence

Protein profiles of six Mycoplasma bovoculi strains obtained from electrophoretic analysis (SDS-PAGE) revealed a close relatedness among these strains. However, the antigenic properties of these proteins varied. Proteins separated on SDS-PAGE gels and transferred to nitrocellulose paper showed strain specific and conserved antigens by western blot analysis when reacted with hyperimmune rabbit serum developed against strain FS8-7 of M. bovoculi. Monoclonal antibodies developed against the same strain recognized a protein band of 94,000 molecular weight found in all strains examined. This band, designated p94 was also detected by sera from calves experimentally exposed to M. bovoculi by ocular instillation. Trypsin treatment of intact M. bovoculi resulted in the removal of this band denoting its proteinaceous nature and membrane surface location. These results indicate that p94 is major protein found on the surface of the plasma membrane of M. bovoculi that is involved in eliciting an antibody response to the organism;The involvement of p94 and protein p29 (29,000 molecular weight) recognized earlier by monoclonal antibodies that block adherence was tested with erythrocytes. Monoclonal antibodies to these surface proteins blocked the attachment of M. bovoculi to erythrocytes. No one antibody blocked the attachment completely indicating that these two proteins are involved in this process and that the organism utilizes at least two receptors for attachment. Characterization of the receptor sites participating in adherence was done by enzymatic treatment. Trypsin treatment of intact M. bovoculi abolished attachment while trypsin or neuraminidase treatment of erythrocytes had no effect. All six strains showed no hemadsorptive or hemagglutinative properties and varied in their hemolytic activity. Hemolysis was seen with mammalian erythrocytes but not with avian erythrocytes;Adherence to bovine lung fibroblasts in vitro and to bovine conjunctival epithelium in vivo was examined by electron microscopy. The organism attached to both cell types without the aid of an apparent specialized structure and possessed no capsule when stained with ruthenium red. Confirmation of the surface location of protein p94 and p29 was done by immunogold labeling. Monoclonal antibody-gold complexes showed widely distributed gold particles around the membrane surface