Serodiagnosis of equine leptospirosis by enzyme-linked immunosorbent assay using four recombinant protein markers

Abstract

Leptospirosis, caused by Leptospira spp., is one of the most common zoonotic diseases in the world. We tested four recombinant proteins of Leptospira interrogans, namely, rLipL21, rLoa22, rLipL32, and rLigACon4-8, to evaluate their potential for use as antigens for the diagnosis of equine leptospirosis. We employed equine sera (n = 130) that were microscopic agglutination test (MAT) negative and sera (n = 176) that were MAT positive for the 5 serovars that most commonly cause equine leptospirosis. The sensitivity and specificity of ELISA compared to MAT were 82.39% and 86.15%, respectively, for LigACon4-8, 77.84% and 92.31%, respectively, for Loa22, 77.84% and 86.15%, respectively, for LipL32, and 84.66% and 83.85%, respectively, for LipL21. When one of the two antigens was test positive, the sensitivity and specificity of ELISA were 93.75% and 78.46%, respectively, for rLigACon4-8 and LipL32, 93.18% and 76.15%, respectively, for rLigACon4-8 and LipL21, 89.77% and 80.77%, respectively, for rLigACon4-8 and Loa22, 91.48% and 78.46%, respectively, for LipL21 and Loa22, 93.75% and 76.92%, respectively, for LipL21 and LipL32, and 90.34% and 80.77%, respectively, for Loa22 and LipL32. In conclusion, we have developed an indirect ELISA utilizing rLigACon4-8, rLoa22, rLipL32, and rLipL21 as diagnostic antigens for equine leptospirosis. The use of four antigens in the ELISA was found to be sensitive and specific, the assay was easy to perform, and the results concurred with the results of the standard Leptospira MAT.

FIG 1

Expression of recombinant proteins. Analysis of affinity chromatography-purified recombinant fragments was performed by Coomassie brilliant blue staining of SDS-PAGE gels. Lane M, protein marker; lane 1, LipL21; lane 2, Loa22; lane 3, LipL32; lane 4, LigACon4-8.

FIG 2

Graphs of IgG ELISA reactivities of 306 equine sera. The x axis indicates the MAT titers of the tested sera. The y axis indicates the ELISA readings (OD₄₅₀).

FIG 3

(A) Western blot results for sera that were MAT negative but ELISA positive. (B) Western blot results for sera that were MAT positive but ELISA negative. The numbers indicate the horse serum numbers.