Research and Development Centre, Indian Immunologicals Limited, Gachibowli, Hyderabad 500 032, India.
J Microbiol Methods. 2012 Sep;90(3):298-304. doi: 10.1016/j.mimet.2012.06.001. Epub 2012 Jun 9.
Bovine tuberculosis (BTB) is a chronic bacterial disease, and a major animal health problem with zoonotic implications. Screening of mycobacterial infections in bovines is traditionally done using the single intradermal tuberculin test. Though the test is widely used, it has its own disadvantages and they include its inability to distinguish between pathogenic and non-pathogenic mycobacterial infections owing to its low specificity. Furthermore, the associated operative difficulties of this test have driven the quest for discovery of new antigens and diagnostic assays leading to the development of the interferon (IFN)- test. Presently, combinatorial testing using the skin test and the interferon gamma assays are being used in the diagnosis of BTB in various control and surveillance programs. In this study, we report the cloning, expression and purification of ESAT-6-CFP-10 fusion protein and its further use in the development of the IFN- gamma ELISPOT assay for accurate diagnosis of BTB in cattle. The BTB diagnosis employing the ELISPOT assay was evaluated using peripheral blood mononuclear cells from culture positive and culture negative cattle. The ELISPOT assay showed higher specificity and sensitivity in detecting BTB when a recombinant ESAT-6-CFP-10 fusion protein was used. The present study indicated that the usefulness of the fusion protein can replace the ESAT-6, CFP-10 or combination of both proteins for detecting BTB in IFN-gamma ELISPOT assay.
牛结核病（BTB）是一种慢性细菌性疾病，是一种具有动物健康问题和人畜共患影响的主要疾病。传统上，使用单一皮内结核菌素试验来筛选牛的分枝杆菌感染。尽管该试验被广泛使用，但它也有其自身的缺点，包括由于特异性低而无法区分致病性和非致病性分枝杆菌感染。此外，由于该试验的操作困难，人们一直在寻求发现新的抗原和诊断检测方法，从而导致干扰素（IFN）试验的发展。目前，在各种控制和监测计划中，使用皮肤试验和干扰素γ检测组合来诊断 BTB。在这项研究中，我们报告了 ESAT-6-CFP-10 融合蛋白的克隆、表达和纯化，及其在 IFN-γ ELISPOT 检测方法的开发中的进一步应用，用于准确诊断牛的 BTB。使用来自培养阳性和培养阴性牛的外周血单核细胞评估了基于 ELISPOT 检测的 BTB 诊断。当使用重组 ESAT-6-CFP-10 融合蛋白时，ELISPOT 检测显示出更高的特异性和敏感性，用于检测 BTB。本研究表明，融合蛋白的有用性可以替代 ESAT-6、CFP-10 或两者的组合，用于 IFN-γ ELISPOT 检测中检测 BTB。
