Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs

Abstract

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

Keywords: canine leptospirosis; diagnosis; enzyme-linked immunosorbent assay; local serovars or isolates; microscopic agglutination test; serological test.

Figure 1

The levels of IgG antibody detected in modified ELISAs against whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP) from the local Thai isolate of Leptospira serovar, including serovar Dadas, at 1:1280 (A), 1:640 (B), and 1:640 (C) sera dilutions. Comparisons among 260 sera from five groups consisting of dogs from Nan Province confirmed as infected via PCR and isolation (Group 1), unvaccinated dogs from Nan Province (Group 2), vaccinated dogs from Bangkok (Group 3), unvaccinated dogs from non-endemic areas (Group 4), and unvaccinated puppies from non-endemic areas (Group 5). The significant differences of the IgG antibody levels between dog sera group were analyzed using Kruskal–Wallis test and Dunn’s post hoc test (p-value < 0.05). (A–C) demonstrated the modified ELISAs against WCP, TMP and OMP from serovar Dadas identified that dogs from endemic area (groups 1 and 2) have the higher level of IgG antibody than dogs in non-endemic areas (groups 3, 4, and 5), with a highly significant difference.

Figure 2

Receiver–operator curve (ROC) and area under the curve of ROC (AUC) of modified ELISAs against whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP) from the local Thai isolates of Leptospira serovars, including serovar Dadas, at 1:1280 (A), 1:640 (B), and 1:640 (C) sera dilutions, with 50 sera from three groups consisting of dogs from Nan Province confirmed as infected via PCR and isolation (Group 1), unvaccinated dogs from Nan Province (Group 2), and vaccinated dogs from Bangkok (Group 3). All the ROC curve and AUC of the ROC of modified ELISAs were analyzed by MedCalc software. (A–C) displayed the sensitivity and specificity of the modified ELISAs against WCP, TMP and OMP from serovar Dadas using the cut-off OD values that were set by ROC analysis showed the moderate sensitivity, specificity and AUC, with p-value < 0.05.