Evaluation of a recombinant Loa22-gold nanoparticle based lateral flow assay for the serodiagnosis of leptospirosis in canine and bovine.

Abstract

Leptospirosis is a globally significant zoonotic disease caused by the pathogenic Leptospira spp. This study aimed to develop a recombinant Loa22 based lateral flow assay for detecting leptospirosis in dogs and cattle. The loa22 gene from Leptospira interrogans was cloned, expressed in E. coli, and purified using Ni-NTA affinity chromatography. The immunoreactivity of the recombinant protein was confirmed by western blotting, and hyperimmune sera against the protein was raised in mice. Gold nanoparticles (AuNP) were conjugated with rLoa22 under optimized conditions and characterized by UV-Vis spectrophotometry, agarose gel electrophoresis, and transmission electron microscopy. The lateral flow assay (LFA) strips were assembled with AuNP-rLoa22 on the conjugate pad and rLoa22 and hyperimmune sera on the test and control lines, respectively. Diagnostic performance of the assay was evaluated using sera from 100 dogs and 102 cattle suspected for leptospirosis and compared to the reference standard microscopic agglutination test (MAT). Six dog and cattle samples each were tested positive by MAT. The developed LFA further tested 06 dog and 07 cattle samples as positive in addition to the MAT positive samples, representing false positives. In dogs, the LFA showed 100% sensitivity and 93.62% specificity, while in cattle, it showed 100% sensitivity (Se) and 92.71% specificity (Sp) with respect to MAT. The n/N for Se and Sp were, Se= 6/6; Sp= 88/94; Se= 6/6; Sp= 89/96. The high observed sensitivity could be attributed to the smaller number of MAT+/LFA + samples that resulted in a wide 95% confidence interval (CI). The test was further validated on another 300 canine and 98 cattle serum samples suspected for leptospirosis (prevalence screens, not a part of the accuracy cohorts). The results underscore the potential of the rLoa22-based LFA as a reliable, rapid diagnostic tool for leptospirosis screening in veterinary settings. Further validation of the study is warranted using samples pre-screened for leptospirosis through other commercially available techniques, including qPCR and ELISA.