Zielinski G C, Ross R F
Veterinary Medical Research Institute, College of Veterinary Medicine, Iowa State University, Ames 50011.
Am J Vet Res. 1993 Aug;54(8):1262-9.
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).(ABSTRACT TRUNCATED AT 250 WORDS)
猪肺炎支原体黏附于猪呼吸道远端黏膜是支原体肺炎发展过程中的一个重要起始事件。一个合适的体外黏附模型对于研究参与此过程的支原体和宿主细胞因子将是有用的。我们已开发出一种黏附试验，使用猪呼吸道纤毛上皮细胞悬液和猪肺炎支原体。通过细胞学刷收集的气管上皮细胞与猪肺炎支原体的肉汤培养物混合，混合物经孵育、稀释、涡旋和沉淀。将沉淀铺在载玻片上，用抗猪肺炎支原体的荧光抗体染色，并通过荧光显微镜评估。荧光主要在纤毛上皮细胞的纤毛簇上观察到。仅观察到少数生物体黏附在纤毛细胞的非纤毛部分或其他细胞类型上。当支原体与从猪肺炎支原体病康复的猪的低稀释度血清预孵育时，黏附受到部分抑制（P<0.05）。当生物体与康复血清的高稀释度、抗猪肺炎支原体的超免疫血清纯化的IgG或含有抗猪肺炎支原体特异性IgA抗体的（来自康复猪的）肺灌洗液低稀释度预孵育时，未观察到黏附的显著抑制。用高碘酸盐和胰蛋白酶对生物体进行预孵育可消除黏附，甲醛可降低黏附（P<0.05），而多种碳水化合物对黏附无影响。用硫酸葡聚糖、硫酸铵、硫酸镁和蛋氨酸预孵育可降低黏附（P<0.05）。用疏水键断裂剂四甲基脲处理细胞 - 支原体混合物，或在无盐条件下孵育，或在低温下孵育也可降低黏附（P<0.05）。（摘要截短于250字）
