Competitive Enzyme-Linked Immunosorbent Assay for Detection of Leptospira interrogans Serovar pomona Antibodies in Bovine Sera

Abstract

A competitive enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (M898) was developed for detection of bovine antibodies to Leptospira interrogans serovar pomona. This assay was evaluated using field sera (n ‫؍‬ 190) with serovar pomona microscopic agglutination test (MAT) titers of >100 as the positive population (group A); field sera (n ‫؍‬ 1,445) which were negative in the MAT (1:100 dilution) for serovar pomona (group B); and sera (from a specific-pathogen-free cattle herd [n ‫؍‬ 210]) which were negative in the MAT (1:100 dilution) for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe (group C). At the cutoff point recommended by receiver operating characteristic (ROC) curve analysis of the combined ELISA results of serum groups A, B, and C, the sensitivity and specificity values were 93.7 and 96.3%, respectively. The value for the area under this ROC curve was 0.977, indicating a high level of accuracy for the ELISA. Similar results were obtained from the analysis of the combined results of serum groups A and B and from the analysis of the combined results of serum groups A and C.In the Canadian cattle population, leptospirosis is predominantly caused by serovar hardjo (now generally accepted as being Leptospira borgpetersenii serovar hardjo type hardjobovis) and serovar pomona (1,6,7,8,9,12,13,14,15). Other serovars such as grippotyphosa and icterohaemorrhagiae have also been detected but at relatively lower levels (6,7,13). Direct detection of these organisms by microscopic examination or culture is impractical due to the low success rate and the amount of time and labor required. Instead, leptospirosis is most often diagnosed serologically with the microscopic agglutination test (MAT) (2). The MAT however, despite its widespread usage and international recognition, is encumbered with a number of limitations. These include the need to use hazardous live bacteria and the amount of time and labor required to test each serum sample against multiple serovars of this organism. In addition, the lack of standard operating procedures and source strains among laboratories and the subjective scoring of results may cause quality assurance difficulties. Due to the drawbacks of the MAT we are developing alternative diagnostic tests for the detection of Leptospira serovars which are of economic importance to Canada.In a previous publication (20), we described two monoclonal antibodies (M897 and M898) that are suitable for incorporation into competitive enzyme-linked immunosorbent assays (ELISAs) for the specific detection of serum antibodies to serovar pomona. In this communication, we report the results of a validation study of a competitive ELISA that was developed with monoclonal antibody M898 for the detection of bovine serovar pomona antibodies. MATERIALS AND METHODSBacterial culture and MAT. The Leptospira organisms were cultured and the MAT was performed as previously described (20).Bovine sera. Field serum samples submitted to Canadian Food Inspection Agency ...

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