RESUMO
BACKGROUND: Streptococcus agalactiae is the main causal pathogen of bovine mastitis (BM), causing considerable economic loss to the dairy industry worldwide. Vaccines against S . agalactiae play an important role in preventing disease. AIMS: The aim of this study was to evaluate the immunoprotection of S. agalactiae pilus island fusion proteins, ancillary protein 1-ancillary protein 2 (AP1-AP2), ancillary protein 1-bone protein (AP1-BP), bone protein-ancillary protein 2 (BP-AP2), and ancillary protein 1-bone protein-ancillary protein 2 (AP1-BP-AP2) in Balb/c mice. METHODS: Four kinds of fusion antigens and the same volume of Freund's complete adjuvant were mixed vigorously to prepare fusion antigen immuno-samples. The mice were immunized 4 times (on the 0th, 7th, 14th, and 28th days) with these samples with an immunizing dose of 50 g/mouse. After the 4th immunization, serology tests were used to evaluate the antibody. The antibody titre produced by AP1-BP-AP2 fusion antigen was the highest, at up to 1:25600. The mice were then injected with 0.5 ml of 2 × 104 CFU/ml clinically isolated S. agalactiae at day 50 and observed daily for the following 7 days. RESULTS: Statistical analyses showed that these 4 kinds of fusion antigens had good protective immunity. Among them, AP1-BP-AP2 fusion antigen had the best protective immunity in Balb/c mice, with an immune protection index (PI) of 80%. Conclusion: This research provides a reliable theoretical basis for screening candidate antigens of the subunit vaccine and detecting antigen preparations of S. agalactiae.
RESUMO
The sip gene encoding for a conserved highly immunogenic surface protein of Streptococcus agalactiae was amplified using polymerase chain reaction (PCR) and subcloned into prokaryotic expression vector pET32a (+) and expressed as a recombinant protein in E. coli BL21 (DE3). An indirect enzyme linked immunosorbent assay (ELISA) was developed using the purified Sip protein as a coating antigen, which could identify S. agalactiae specific antibody in sera. The coating antigen at a concentration of 3.125 µg/ml, serum diluted to 1:160, and HRP-conjugated secondary antibody concentration at 1:4000 was found to be most effective in exhibiting positive result. The ELISA was found to be highly specific for S. agalactiae that may be used for the detection of the pathogen in mastitis cases, for epidemiological studies and for surveillance.
