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.

Detection of intraocular leptospiral DNA, antibodies and Leptospira spp. in horses with equine recurrent uveitis in different laboratories

“…The prevalence of leptospiral antibodies usually measured via the MAT varies greatly in horses in a multitude of serological surveys. The seroprevalence of leptospiral antibodies ranged between 5 and 95 % in horses, depending on the age and the geographic location of the residence of the horse (Kitson-Piggot et al 1987, Lees et al 1994, Rocha et al 2004, Ye et al 2014). Usually titers ≥ 1:100 were rated as positive, only Kitson-Piggot et al (1987) rated a titer ≥ 1:80 as positive, but only 5 % of the three year old horses showed a positive result.…”

“…Since the MAT results need to be regarded carefully for interpretation, as outlined above, the ELISA is an improper test to safely identify leptospiral infection in equine patients. A higher sensitivity of the ELISA can be achieved by combining multiple recombinant proteins of a specific serovar in one test (Ye et al 2014). …”

Molecular characterisation of Leptospira strains in Pakistan

“…Its clinical signs are nonspecific, making its diagnosis a challenge (29). Despite high rates of seropositivity among animals in Pakistan (2,20), to date, there have been no data on the molecular studies of leptospirosis in horses or in any mammalian host.…”

Leptospiral major outer membrane protein

“…Among a total of 86 MAT-positive blood samples, all were positive with the LipL32-ELISA method [43]. A combination of four antigens rLipL21, rLoa22, rLipL32 and rLigACon4-8 was used by Ye et al [44] to detect leptospirosis in horses. Use of four antigens in ELISA increased the sensitivity and specificity of serodiagnosis of leptospirosis in horses and the results were comparable to the MAT results [44].…”