Development of an Enzyme-Linked Immunosorbent Assay and Co-Agglutination Test for Diagnosis of Leptospirosis

Abstract

An enzyme-linked immunosorbent assay (ELISA) for measuring leptospiral antibodies was developed. Desoxycholate extracted and type specific antigens of Leptospira interrogans serovars hardjo, szwajizak, pomona and grippotyphosa and sera from experimentally infected rabbits were used. No significant titer difference was observed when either horseradish peroxidase (HP) or alkaline phosphatase (AP) was used to label the anti immunoglobulin, although HP gave more sensitive reaction for early sera. When compared with the microscopic agglutination (MA) test, the ELISA with desoxycholate extracted antigens was more sensitive but showed more cross reactions. Type specific antigen of hardjo gave 80% sensitivity and 96.5% specificity in differentiating hardjo antibodies from that of other serovars in rabbits. The ELISA, therefore, should include the use of type specific antigens and HP-conjugated anti species immunoglobulin.

Results of the ELISA in goats experimentally infected with hardjo and szwajizak indicated the possibility of the use of this species to differentiate hardjo from szwajizak isolates.

In experimentally infected cattle, the ELISA was more sensitive than the MA. The assay detected serological responses of cattle vaccinated with leptospiral bacterins, which were not detectable by the MA test. In screening naturally infected cattle, the ELISA gave between 57.5-72.5% agreement with the MA test. Since type specific antigens of hardjo and szwajizak still cross reacted to each other with cattle sera, further purification of the antigens is necessary.

Since only 4 out of 11 immune rabbit sera were positive in the co-agglutination test, further studies are necessary.