Detection of canine IgG and IgM antibodies to Coccidioides immitis using a novel enzyme-linked immunosorbent assay (EIA) method

Accurate diagnosis of coccidioidomycosis in the canine is essential in veterinary medicine. A positive diagnosis of coccidioidomycosis carries serious consequences, from expensive and prolonged treatment to euthanasia. Detection of antibodies to Coccidioides immitis via complement fixation has been the gold standard assay for serologic diagnosis of coccidioidomycosis. The presence of anticomplementary factors in symptomatic dogs obviates the usefulness of this test. The agar gel immunodiffusion (AGID) method is sensitive and specific, although it requires a long turnaround time. The goal of this study was to develop a sensitive and specific assay capable of detecting canine IgG and IgM antibodies to Coccidioides immitis utilizing an enzyme-linked immunosolvent assay (EIA). The turnaround time of the EIA test vs. AGID was also evaluated. The EIA method was compared to traditional ACID methods measuring the presence of IgG and IgM antibody as it reacts with CF and TP antigens, respectively. Serum samples from symptomatic dogs suspected of having acute or chronic coccidioidomycosis were submitted to our laboratory over a 3-month period. Microwells coated with purified CF and TP antigens, manufactured by Meridian Diagnostics, Inc., were utilized to test for IgG and IgM specific antibodies, respectively. Anti-canine IgC specific and IgM specific antibodies, manufactured by Organon Teknika-Cappel, Durham, NC, were then utilized to complete the sandwich in a standard EIA format. Reaction absorbances were read at 450nm and 630nm on a standard spectrophotometer; manufacturer's recommended cutoffs were adhered to for the determination of negative, indeterminate, and positive interpretations. Compared to the AGID method, which is assumed to be 100 percent, EIA's sensitivity and specificity for IgG were 99.4 percent and 91.8 percent, respectively, Sensitivity and specificity for detection of IgM were 69.6 percent and 95.6 percent, respectively, The lower sensitivity of IgM detection in the EIA method may be attribustable to the known reactivity of IgG to the TP antigen, Turnaround time decreased from up to 5 days for the AGID, to less than 1 day with EIA. The EIA method appears to be a sensitive, specific, and clinically useful assay for the serological diagnosis of coccidioidomycosis in the canine.