Wawegama Nadeeka K, Browning Glenn F, Kanci Anna, Marenda Marc S, Markham Philip F
Asia-Pacific Centre for Animal Health, Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia.
Clin Vaccine Immunol. 2014 Feb;21(2):196-202. doi: 10.1128/CVI.00670-13. Epub 2013 Dec 11.
Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.
牛支原体可引发牛的一系列疾病，包括乳腺炎、关节炎和肺炎。然而，对感染进行准确的血清学诊断仍然存在问题。本文所述研究旨在鉴定一种可用于开发更特异、灵敏诊断检测方法的抗原。在实验感染牛支原体的犊牛血清抗体的蛋白质印迹法中，始终检测到一种226 kDa的免疫原性蛋白。该蛋白被证明是一种具有脂肪酶活性的膜蛋白，被命名为支原体免疫原性脂肪酶A（MilA）。MilA的不同区域在大肠杆菌中作为谷胱甘肽S-转移酶（GST）融合蛋白表达，并且显示来自氨基末端的重组产物与牛支原体特异性牛血清具有强免疫反应性。免疫反应性最强的融合蛋白GST-MilA-ab被用于开发间接IgM和IgG酶联免疫吸附测定（ELISA）。IgM ELISA在感染后2周检测牛支原体特异性IgM抗体，灵敏度为97.1%，特异性为63.3%，而IgG ELISA在感染后3周检测牛支原体特异性IgG，灵敏度为92.86%，特异性为98.7%，表明IgG ELISA有潜力作为检测牛感染的灵敏且特异的检测方法。
