Adherence of Mycoplasma hyopneumoniae to porcine ciliated respiratory tract cells

Abstract

Summary Adherence of Mycoplasma hyopneumoniae to the mucosa of the distal portion of the respiratory tract of swine is an important initial event in development of mycoplasmal pneumonia. A suitable in vitro model of adherence would be useful for investigation of mycoplasmal and host cell factors involved in this process. We have developed an adherence assay, using suspensions of porcine respiratory tract ciliated epithelial cells and M hyopneumoniae . Tracheal epithelial cells, collected by use of cytologic brushes, were mixed with broth cultures of M hyopneumoniae and the mixtures were incubated, diluted, vortexed, and sedimented. Pellets were spread on glass slides, stained with a fluorescent antibody against M hyopneumoniae , and evaluated by fluorescent microscopy. Fluorescence was observed principally among cilia on the ciliated tufts of epithelial cells. Only a few organisms were observed adhering on the nonciliated parts of ciliated cells or on other cell types. When mycoplasmas were preincubated with low dilutions of serum from swine convalescing from M hyopneumoniae disease, attachment was partially inhibited ( P < 0.05). Significant inhibition of attachment was not observed when organisms were preincubated with higher dilutions of convalescent serum, with purified IgG from hyperimmune serum against M hyopneumoniae , or with low dilutions of lung lavage fluids (from convalescent swine) that contained specific IgA antibodies against M hyopneumoniae . Preincubation of the organisms with periodate and trypsin abolished attachment and formaldehyde decreased it ( P < 0.05), whereas a variety of carbohydrates had no effect on attachment. Preincubation with dextran sulfate, ammonium sulfate, magnesium sulfate, and methionine reduced attachment ( P < 0.05). Treatment of cell- Mycoplasma mixtures with the hydrophobic bond-breaking agent tetramethylurea, or incubation in absence of salt, or at low temperature also reduced attachment ( P < 0.05). Attachment was not observed when ovine, rabbit, or guinea pig ciliated respiratory tract cells were mixed with M hyopneumoniae . Attachment of M dispar to porcine ciliated cells could not be detected, whereas M hyorhinis attached nonspecifically to all cell types in suspensions of porcine tracheal mucosa. These results indicate that adherence of M hyopneumoniae may be a host-specific event that is mediated by proteins and carbohydrates on the surface of the organism and by sulfur-containing molecules in the host cell membrane. The highly polarized location of the mycoplasmas on the cilia of epithelial cells indicates possible existence of stereospecific interactions between mycoplasmal adhesin(s) and receptor(s) on host cells. However, decreased adherence obtained by incubating Mycoplasma -ciliated cell mixtures with tetramethyl-urea, by incubating mixtures in absence of salt or at low temperature, indicates that nonspecific hydrophobic interactions might have a role in the attachment process.