The Establishment Of The Mouse Evaluation Model For Brucella S2 Vaccine And The Screening Of Differential Diagnostic Antigens

Brucellosis is a wide spread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating, febrile illness in humans. Brucella abortus, Brucella melitensis and Brucella suis are the major pathogenic species to humans. Vaccination with live attenuated Brucella suis strain 2 (S2) vaccine is an essential and critical component in the control of brucellosis in China. The S2 vaccine is very effective in preventing brucellosis in goats, sheep, cattle and swine. However, there are still debates outside of China whether the S2 vaccine is able to provide protection against heterologous virulent Brucella species. We investigated the residual virulence, immunogenicity and protective efficacy of the S2 vaccine in BALB/c mice by determining bacteria persistence in spleen, serum antibody response, cellular immune response and protection against a heterologous virulent challenge. The S2 vaccine was of low virulence as there were no bacteria recovered in spleen four weeks post vaccination. The vaccinated mice developed Brucella-specific IgG in 2-3 weeks, and a burst production of IFN-y at one week as well as a two-fold increase in TNF-a production. The S2 vaccine protected mice from a virulent challenge by B. melitensis M28, B. abortus 2308 and B. suis S1330, and the S2 vaccinated mice did not develop any clinical signs or tissue damage. Our study demonstrated that the S2 vaccine is of low virulence, stimulates good humoral and cellular immunity and protects animals against infection by heterologous, virulent Brucella species. Our studies provided data basis of animal model for the usage of S2 by OIE.Because S2 and the wild virulent strains are smooth strains, the biggest problem in brucellosis control is unable to distinguish the vaccine immune antibody and the natural infection antibody. We screened the differential diagnostic antigens in S2 membrane proteins by immunoprotoemics. The S2 membrane proteins were separated by two dimensional electrophoresis, and the gels were blotted with 3 kinds of mixed sera from 30 negative, S2 vaccinated and natural infected sheep, respectively. Total 113 protein spots with differential immune-reactivity were identified and 30 protein spot with largest difference were selected for MALDI-TOF MS analysis and bioinformatics analysis.14 proteins were identified and the bioinformatics analysis showed that the 14 proteins exercise of 13 kinds of molecular function, participate in the composition of 6 cell component, and participate in 13 different biological process. Also, immunoprecipitation (IP) were carried out to capture the binding proteins with the three kinds of mixed sera from S2 membrane proteins. The IP conjugates were identified by Q-Exactive MS analysis, and total 182 candidate diagnostic targets were obtained. The bioinformatics analysis showed that the 182 proteins exercise 129 kinds of molecular function, participate in the composition of 91 cellular component, and participate in 137 different biological process. Comparing the two methods,12 proteins were identified by both methods. According to the function of the identified proteins, total 10 proteins (1#-10#) are expected to become the differential diagnostic antigen, which 2 proteins were obtained by 2DE&Western-blot&MALDI-TOF and 10 proteins were obtained by IP&QE (2 proteins repeated by the 2DE&Western-blot&MALDI-TOF method).In order to verify the role of the 10 candidates in the differential diagnosis, the genes were amplified and the proteins were expressed with a GST-tag in E. coli.8 proteins except 1# and 5# were expressed after induction. The expressed proteins were detected by Western-blot and ELISA by the 3 kinds of mixed sera, and the 2# (universal stress protein),3# (glycoside hydrolase family 43) and 10# (hypothetical protein) were screened out to have differential diagnostic effect. The three proteins were then purified by GST affinity chromatography. Using the purified proteins as the coated antigen, the ELSIA parameters were optimized by the matrix test and the single factor method. Then, the 30 sera from infected sheep and 656 sera from S2 vaccinated sheep were tested using the established ELISA method. The sample determinative ratio for infected samples was about 80% and 60-70% for ELISA with 2# and 3# and 10# as the coated antigen, respectively. For the sera from S2 vaccinated sheep, a small amount was detected as the natural infected samples. Due to the established differential ELSA method lacking of the sensitivity and specificity temporarily, further verification is needed to test a large number of infection samples with clear background. Our study provides the basis of resolution for the differential diagnosis problem of brucellosis and contributes to the clearance of brucellosis.