ICAR-National Institute of Veterinary Epidemiology and Disease informatics (NIVEDI), Yelahanka, Bengaluru, Karnataka, India.
Jain University, Bengaluru, Karnataka, India.
Lett Appl Microbiol. 2021 Jun;72(6):730-740. doi: 10.1111/lam.13461. Epub 2021 Mar 4.
Leptospirosis is a re-emerging zoonotic disease of animals and humans caused by pathogenic Leptospira, which has major public health concerns. The study is aimed to express the recombinant outer membrane protein (OMP) A-like protein (rLoa22) and transmembrane (rOmpL37) protein of Leptospira interrogans serovar Hardjo in the Escherichia coli and their evaluation as a diagnostic antigen in the latex agglutination test (LAT) to detect anti-leptospiral antibodies in the sera of animals. The Loa22 and OmpL37 genes lacking signal peptide coding sequences were individually amplified (522 and 963 bp), by polymerase chain reaction, and directionally cloned into a pETite N-His Kan vector for expression. The expressed purified proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblot, which confirmed leptospiral specific reactive protein with a molecular weight of ~19 and 36 kDa, respectively. The sensitized latex beads coated with these OM proteins separately were evaluated in LAT using cattle sera of microscopic agglutination test (MAT) confirmed positive (n = 53) and negative (n = 52) cases of leptospirosis. The rLoa22 LAT and rOmpL37 LAT revealed the relative diagnostic sensitivity of 94·34 and 96·23%, diagnostic specificity of 92·31 and 96·15% and accuracy of 93·33 and 96·19%, with the excellent agreement of Cohen's kappa value of 0·87 and 0·92, respectively. After extensive evaluation, this rapid recombinant protein-based field diagnostic test can be applied as a screening test for the detection of anti-leptospiral antibodies in the sera of animals in the field conditions.
钩端螺旋体病是一种由致病性钩端螺旋体引起的动物和人类重新出现的人畜共患疾病，具有重大的公共卫生关注。本研究旨在表达钩端螺旋体血清型硬蜱的重组外膜蛋白（OMP）A 样蛋白（rLoa22）和跨膜（rOmpL37）蛋白在大肠杆菌中，并将其作为乳胶凝集试验（LAT）中的诊断抗原进行评估，以检测动物血清中的抗钩端螺旋体抗体。通过聚合酶链反应分别扩增（522 和 963 bp）缺乏信号肽编码序列的 Loa22 和 OmpL37 基因，并定向克隆到 pETite N-His Kan 载体中进行表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹对表达的纯化蛋白进行了特征分析，分别证实了具有约 19 和 36 kDa 分子量的钩端螺旋体特异性反应蛋白。分别用这些 OM 蛋白敏化的乳胶珠在 LAT 中进行了评估，使用经过显微镜凝集试验（MAT）证实的阳性（n = 53）和阴性（n = 52）牛血清进行了检测。rLoa22 LAT 和 rOmpL37 LAT 分别显示相对诊断灵敏度为 94.34%和 96.23%、诊断特异性为 92.31%和 96.15%和准确性为 93.33%和 96.19%，Cohen's kappa 值的优异一致性分别为 0.87 和 0.92。经过广泛评估，这种快速的基于重组蛋白的现场诊断测试可作为现场条件下检测动物血清中抗钩端螺旋体抗体的筛选测试。
