Immunochemical characterization of antigens of Brucella canis and their use in seroprevalence study of canine brucellosis.

Abstract

OBJECTIVE: To explore immunochemical characterization of antigens of Brucella canis (B. canis), and the use in seroprevalence study of canine brucellosis. METHODS: External hot phosphate buffer saline extract (HPBSE) and internal sonicated (SA) antigens were prepared from B. canis strain MEX 51 and immunochemically characterized. These antigens were used to test 527 serum samples of dogs by 2-mercaptoethanol-tube agglutination test (2 ME-TAT), agar gel immunodiffusion test (AGID), dot-ELISA and indirect enzyme-linked immunosorbent assay (I-ELISA) to assess the seroprevalence of canine brucellosis. RESULTS: The protein content of HPBSE and SA antigens was 0.387 mg/mL and 0.195 mg/mL, respectively, whereas carbohydrate content was 0.174 mg/mL and 0.150 mg/mL, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5%) of HPBSE and SA, revealed 6 and 8 visible peptide bands ranging from 18-80 kDa and 12-45 kDa, respectively. Western blot analysis showed immunodominant bands of MW 12, 28, 39 and 45 kDa for HPBSE and 20-24 kDa for SA. The AGID revealed HPBSE as more specific antigen than SA but both I-ELISA and dot-ELISA indicated SA antigen to be more specific and reliable than HPBSE. The seroprevalence of canine brucellosis was 2.27% by 2ME-TAT, 1.5% by AGID, 3.03% by dot-ELISA and 16.12% by I-ELISA. CONCLUSIONS: On the basis of the results of present study, we concluded that HPBSE is suitable antigen for AGID, which is more specific; whereas SA antigen is suitable for I-ELISA, which is highly sensitive. Therefore, initial screening of serum samples should be carried out by I-ELISA followed by confirmation with AGID.