Zielinski G C, Young T, Ross R F, Rosenbusch R F
Veterinary Medical Research Institute, College of Veterinary Medicine, Iowa State University, Ames 50011.
Am J Vet Res. 1990 Mar;51(3):339-43.
This work was an attempt to develop an in vitro adherence model for Mycoplasma hyopneumoniae, using monolayers of human and porcine lung fibroblasts and porcine kidney cells. Mycoplasma hyopneumoniae grown in Friis mycoplasma broth was radiolabeled with 35[S]-methionine, washed, concentrated, and inoculated on the monolayers. After 15 minutes of centrifugation to facilitate adherence, monolayers were washed 3 times, dissolved with 0.1N NaOH, and suspended in scintillation liquid, and the radioactivity was determined in a liquid scintillation counter. Adherence, measured as a percentage of counts added, varied according to the mycoplasma strain and the cell line used. Comparison of strains J, 144L, and 232 of M hyopneumoniae revealed 7.5 +/- 5.9, 31.9 +/- 13, and 9.6 +/- 5% adherence to porcine kidney cells, respectively. Slightly different, but proportionally the same relationships were obtained with swine or human fibroblasts. Adherence was decreased slightly by repeated washings of the mycoplasma-treated cell monolayers; however, a plateau was reached, indicating irreversibility of the adherence process. Pretreatment of cell monolayers with nonlabeled organisms substantially blocked adherence by labeled organisms. Dilution of labeled organisms resulted in an increased proportion adhering. Therefore, it appears that the adherence was a receptor-dependent event. Treatment of the mycoplasmas with trypsin prior to the inoculation of monolayers resulted in a marked reduction in adherence. Treatment of the mycoplasmas with hyperimmune swine serum against M hyopneumoniae or normal swine serum resulted in 80 to 90% reduction of adherence; however, no inhibition occurred when mycoplasmas were treated with purified IgG from the hyperimmune serum.
本研究旨在利用人及猪肺成纤维细胞单层和猪肾细胞建立猪肺炎支原体的体外黏附模型。在弗里斯支原体肉汤中培养的猪肺炎支原体用³⁵[S]-甲硫氨酸进行放射性标记，洗涤、浓缩后接种于细胞单层。经15分钟离心以促进黏附后，细胞单层洗涤3次，用0.1N氢氧化钠溶解，悬浮于闪烁液中，在液体闪烁计数器中测定放射性。以添加计数的百分比衡量的黏附情况因支原体菌株和所用细胞系而异。对猪肺炎支原体的J、144L和232菌株进行比较发现，它们对猪肾细胞的黏附率分别为7.5±5.9%、31.9±13%和9.6±5%。用猪或人成纤维细胞获得的结果略有不同，但比例关系相同。对经支原体处理的细胞单层进行反复洗涤会使黏附略有降低；然而，会达到一个平稳期，表明黏附过程是不可逆的。用未标记的支原体预处理细胞单层可显著阻断标记支原体的黏附。标记支原体的稀释导致黏附比例增加。因此，看来黏附是一个依赖受体的事件。在接种细胞单层之前用胰蛋白酶处理支原体导致黏附显著降低。用抗猪肺炎支原体的超免疫猪血清或正常猪血清处理支原体导致黏附降低80%至90%；然而，用超免疫血清中的纯化IgG处理支原体时未出现抑制作用。
