Development and evaluation of two ELISA tests for diagnosis of canine brucellosis

Abstract

Canine brucellosis is a disease that in most cases causes symptoms ranging from mild to severe reproductive disorders. In most infected animals though, the disease is inapparent, making clinical diagnosis very difficult. The only method by which a diagnosis can be made with certainty is the isolation of Brucella canis. When serological tests are performed, a large number of false positive results occur due to the presence of antigenic surface determinants common to B. canis and other bacteria. Blood sera from 225 dogs were tested using the 2-ME TAT method and in-house ELISA tests using antigen extracts obtained by thermal extraction and by ultrasonication. Electrophoretic analysis and densitometric quantification of the antigen showed that in the thermally-extracted antigen, the most abundant molecular mass fraction was 10.95 kDa with a proportion of 43.12 %, which corresponded to the R-LPS of Brucella. The same fraction was present in the ultrasonicated extract, but accounted for 11.56 % of the protein mass, i.e., 3.7 times lower than in the thermal extraction. The protein composition of the ultrasonicated antigen was significantly richer than in the thermal extraction, and the quantitative composition was dominated by proteins with molecular masses of 30.5, 24.5, 38.0 and 22.0 kDa, which belonged to the outer membrane proteins. In the ELISA test with the thermally-derived antigen, 44 (19.55 %) of 225 sera tested positive, while in the ELISA test with the ultrasonicated antigen, 37 (16.44 %) were positive. In addition to the 2-ME TAT, the use of an indirect ELISA test performed on ultrasonically extracted antigen is recommended for serological testing in order to make an as accurate as possible diagnosis.