title: A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis.
abstract: The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the 'Bruce ladder' multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China. Electronic address: wjingyu2004@126.com.
authors.forename: Jing-Yu
authors.initials: JY
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Wu
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors.forename: Wan-Hua
authors.initials: WH
authors.lastname: Liu
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors.forename: Juan-Juan
authors.initials: JJ
authors.lastname: Ren
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors.forename: Pan
authors.initials: P
authors.lastname: Tang
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
authors.forename: Yuan-Hao
authors.initials: YH
authors.lastname: Qiu
authors.affiliation: Department of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan.
authors.forename: Chi-Young
authors.initials: CY
authors.lastname: Wang
authors.affiliation: Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan.
authors.forename: Ching-Dong
authors.initials: CD
authors.lastname: Chang
authors.affiliation: Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan; Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan; Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung 402, Taiwan. Electronic address: hjliu5257@nchu.edu.tw.
authors.forename: Hung-Jen
authors.initials: HJ
authors.lastname: Liu
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000276:immunology; D015153:Blotting, Western; D002003:Brucella abortus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D017347:Brucella melitensis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D041021:Brucella suis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002006:Brucellosis / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D013045:Species Specificity
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001428:Bacterial Vaccines
keywords: B. suis S2; Brucellosis; Cattle; Differential diagnosis; Indirect ELISA; repA-related protein