Preparation and application of standard positive serum of Brucella canis

Abstract:

[Background] Brucella canis, one of the six representative species of Brucella, is a zoonotic pathogen that poses a serious threat to the health of dogs. [Objective] To prepare the B. canis positive serum, providing the reference material for the development and application of diagnostic reagents for canine brucellosis. [Methods] Beagle dogs were immunized multiple times with the inactivated bacterial suspension (approximately 1.5×10¹⁰ CFU/mL) to obtain the positive serum of B. canis. By conducting the serum agglutination test, we calibrated the serum titer with the international standard serum provided by the European Union. We then used the standard positive serum prepared in this study as the reference material for quality control of the detection sensitivity of the B. canis antigen for rose bengal test and the B. canis indirect ELISA antibody detection kit developed by our laboratory. Furthermore, we used the above two methods to detect the B. canis antibody in the serum samples of pet dogs in Beijing and analyzed the coincidence rates of results between the two methods and the complement fixation test. [Results] The results of the serum agglutination test showed that the titer of B. canis positive serum was 2 400 IU/mL. The serum was diluted with the negative dog serum to reach a titer of 1 000 IU/mL, subpackaged, and freeze-dried to give the standard positive serum of B. canis. With the standard positive serum for quality control, the detection sensitivities of the B. canis antigen for rose bengal test and the B. canis indirect ELISA antibody detection kit developed by our laboratory were both 20 IU/mL. The positive rates obtained by both the B. canis antigen for rose bengal test and the complement fixation test was 6.25% and that obtained by the B. canis indirect ELISA antibody detection kit was 5.97%. The coincidence rates of results between the two detection methods and the complement fixation test were 94.31% and 94.60%, respectively. [Conclusion] The standard positive serum of B. canis that we prepared in this study provides a quality control sample for the development of diagnostic reagents for B. canis.

Key words:Brucella canis; positive serum;calibration of titer;serum agglutination test;indirect ELISA