| Bovine brucellosis and bovine tuberculosis are the two most important zoonotic infectious diseases of cattle. Brucellosis is caused by Brucella abortus, which may leads to human chronic infections, ruminants infertility and abortion, therefore threats human health and the development of cattle industry. Bovine tuberculosis is caused by Mycobacterium bovis (M.bovis), which is also an important cause of human tuberculosis. According to state regulation, every year bovine brucellosis and bovine tuberculosis should be screened twice an year, in Spring and Fall. The detection methods of brucellosis are mainly based upon detection of serum antibodies to lipopolysaccharide (LPS) O chain. The method for detecting bovine tuberculosis is tuberculin skin test (TST). The traditional detection methods for these two diseases are time consuming and laborious, in addition, there is non-specific reaction between these two methods and other methods. The purpose of this study was to establish a double colloidal gold immunochromatographic assay (GICA) detection method to detect both bovine brucellosis and bovine tuberculosis with high specificity and sensitivity, with more simple execution.The traditional Brucella serologic assay (e.g., the complement fixation test and the seroagglutination test) are mainly based on the detection of antibodies directed against the lipopolysaccharide (LPS). This dominant antigen is shared both by vaccinal and virulent Brucella. Therefore, the differentiation between infection and vaccination is very difficult. Moreover, tests based on anti-LPS antibodies have extensive cross-reactions with other gram-negative bacteria. Some papers have reported that some Brucella proteins such as p17, Omp25 and bp26 are able to overcome the drawbacks mentioned above and could be the candidate for diagnostic antigen in brucellosis detection.Streptococcal Protein G (SPG) is located in the cell wall of Streptococcus A, C and G group, can bind to Fc domain of IgG. Due to the merit of SPG, it is widely used as the IgG capture protein.Our laboratory screened these three diagnostic antigens and developed Colloidal GICA method. Based on the above background, in this study a colloidal gold immunochromatographic strip which can detect bovine tuberculosis and bovine brucellosis simultaneously was established. In the GICA assay, SPG was used to bind to gold particles Esat-6, CFP-10 and Rv3872 fusion protein (RCE), as bovine tuberculosis diagnostic antigen. The p17, Omp25 and bp26 were compared for brucellosis tests, bp26 was selected as bovine brucellosis diagnostic antigens. The main contents and results are as follows:1. Clonning, expression, and immunogenicity analysis of the recombinant protein pl7, Omp25 and bp26.Using the genome of B. melitensis reference strain as the template, the p17, Omp25 and bp26 genes were amplified with PCR and the PCR product was cloned into to pET-30a plasmid vector. The resultant recombinant plasmid was sequenced and the correct recombinant plasmid was transformed into E.coli BL21. The insert genes were induced to be expressed in BL21. SDS-PAGE results showed that both p17 and bp26 were expressed in soluble form, while Omp25 was expressed in an insoluble form. Western bloting assay was performed to detect the immunogenicity of these three recombinant proteins, using bovine serum as the first antibody, the results showed that all the three recombinant protein are immunogenic. By coating the three recombinant proteins separately to microtiter plates 50ng/well, an iELISA was established. The iELISA was used to detect 44 bovine serum (24 positive samples,20 negative samples), and the results were compared to those of IDEXX brucella antibody detection kit. The results demonstrated that bp26 was the best as an antigen to test serum antibody to brucella.2. Clonning and, expression of the recombinant SPGThe SPG fragment which includes 4 copies of the strongest IgG binding domain C3 and D domain (incluing 15 amino acids) as the linker which was inserted between each two C3 was artificially synthesized by the commercial company. The fragment was designated as SPG-(C3)4(D)3, and SPG-(C3)4(D)3(Cys)2 by adding 2 cysteins at C terminal. These two synthetic sequences were cloned into the pET-30a, transformed into E.coli BL21 and expressed after induction. SDS-PAGE analysis showed that both the recombinant proteins were successfully expressed in a soluble form. The Western blot assay results indicated these two proteins are able to bind bovine IgG very well, and capacity of SPG-(C3)4(D)3(Cys)2 to bind IgG was not interfered by Cys.3. Development of a novel immunochromatographic strip for rapid detection of antibodies against bovine tuberculosisBy employing MPB83 as a gold colloidal binding protein, the SPG-(C3)4(D)3 as coated protein at the test line, rabbit anti-MPB83 polyantibody as the coated protein at the quality control line, an immunochromatographic trip was developed for rapid detection of antibodies against bovine tuberculosis. Compared the commercial strip to detect antibodies to MPB70/MPB83, the sensitivity of this new strip was improved dramatically, meanwhile, the specificity kept consistent with the MPB70/MPB83 strip, indicating that SPG-(C3)4(D)3 served as IgG binding protein can significantly improve the strip sensitivity.4. Development of a novel immunochromatographic strips for rapid dual detection of antibodies against bovine brucellosis and tuberculosisSPG-(C3)4(D)3(Cys)2 was used as the gold colloidal binding protein. The optimal labeling amount of SPG-(C3)4(D)3(Cys)2 was 3.25ug/mL. The bp26 was coated on the nitrocellulose and served as the diagnostic antigen for brucellosis, while Rv3872-CFP10-Esat6 (RCE) was served as the diagnostic antigen for bovine tuberculosis, and rabbit IgG as the control line protein. The optimal coating concentrations of bp26, RCE, rabbit IgG was 2.0 mg/mL,2.5 mg/mL and 10mg/mL, respectively. The components were assembled and the immunochromatographic strips for dual rapid detection of antibodies against bovine brucellosis and tuberculosis was developed.In total,80 sera samples (40 brucellosis positive samples,40 brucellosis negative samples; 13 Bovine tuberculosis positive samples,67 Bovine tuberculosis negative samples; 7 double positive samples) were tested using immunochromatographic strips, and the brucellosis detection results were compared with the rose-bengalplate agglutination test and IDEXX brucella antibody detection kit, this show that the sensitivity was 87.5%(35/40), specificity was 92.5%(37/40), and accuracy was 90% (72/80); the bovine tuberculosis detection results were compared with the ELIS A kit to detect the Antibodies against Mycobacterium bovis, the sensitivity was 84.6%(11/13), specificity was 89.5%(60/67), and accuracy was 88.7%(71/80)... |
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