An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars

Abstract

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G1 that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay. At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation.

Figure 1

Frequency distribution of enzyme linked immunosorbent assay (ELISA) results of (A) 601 microscopic agglutination test (MAT)-positive field sera and (B) 3107 MAT-negative field sera. The x-axis shows high positive control (%) values and the y-axis shows the number of sera.

Figure 2

Receiver operating characteristic (ROC) curve obtained from the analysis of the enzyme linked immunosorbent assay (ELISA) results of 601 microscopic agglutination test (MAT)-positive and 3107 MAT-negative field sera. The false-positive rate (100-specificity [x-axis]) is plotted against the true-positive rate (sensitivity [y-axis]) for each high positive control (HPC) (%) cut-off point applied. An optimal cut-off point of ≥ 42% HPC is indicated (arrow). The area under the ROC curve = 0.977.

Figure 3

Enzyme linked immunosorbent assay (ELISA) results of sera obtained from serial bleedings of cattle experimentally infected with hardjobovis (H1 and H2), canicola (Ca), copenhageni (Co), and grippotyphosa (G). The x-axis shows time (weeks post-inoculation) and the y-axis shows percent high positive control (HPC) value. A cut-off value of ≥ 42% HPC is used to distinguish between positive and negative results.

Figure 4

Quality assurance data obtained for the enzyme linked immunosorbent assay (ELISA). The y-axis shows the mean high positive control (%) values obtained for 4 (P = pomona serum, Co = copenhageni serum, N = microscopic agglutination test [MAT]-negative serum, and B = buffer) of the 6 quality controls reagents that are included in every ELISA plate. Upper and lower limits (± 2 s, respectively) for each control are indicated. The x-axis shows assay number of the 51 plates included in this analysis.