A new Brucella canis species-specific PCR assay for the diagnosis of canine brucellosis.

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis. The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6pg/µl by DNA dilution, or 3×10(3) colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis. Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis, and the new assay will provide a valuable tool for the diagnosis of B. canis infection.

Immunoproteomics of Brucella abortus RB51 as candidate antigens in serological diagnosis of brucellosis.

The current brucellosis serodiagnostic assays are chiefly based on detecting anti-LPS (lipopolysaccharide) antibodies. However, cross-reaction with some gram-negative bacteria can occasionally induce due to similar O-polysaccharide (OPS) structure. Therefore, the aim of the present study was to identify new candidate antigens from Brucella abortus RB51, a mutant strain lacking the LPS portion, which might be valuable in brucellosis diagnosis. To detect potential antigens, immobilized pH gradients (IPG) strips with three ranges (pH 3-5.6, 4-7 and 6-11) were applied. After separating the insoluble proteins of B. abortus RB51 using two-dimensional electrophoresis (2-DE), their immunogenicity was evaluated by western blotting using four types of antisera - B. abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7-positive, and B. abortus-negative bovine sera. Among the several immunogenic spots, the spots showing specific reactivity with only the B. abortus-positive antisera, were considered as candidate antigens. Overall, eleven immuno-reactive proteins were identified, as follows: Cu/Zn superoxide dismutase, histidinol dehydrogenase, chaperonin DnaK, chaperonin GroES, beta-ketoadipyl CoA thiolase, two-component response regulator, the cell-division protein FtsZ, aldehyde dehydrogenase, 50s ribosomal protein L10 and invasion protein B. These selected highly immunogenic protein spots might be useful as alternative antigens for brucellosis and helpful in reducing the cross-reactivity.

Epidemiologic relatedness between Brucella abortus isolates from livestock and wildlife in South Korea.

To investigate the epidemiologic relatedness of Brucella abortus isolates from Chinese water deer (Hydropotes inermis) and goral (Naemorhedus goral raddeanus) in 2010-2011, 22l isolates from livestock (including domestic elk, Cervus canadensis) were analyzed using the multilocus variable-number tandem repeats analysis. In the clustering analysis, Korean B. abortus isolates were divided into 40 genotypes by 18 markers, and 2 B. abortus isolates from wildlife were clustered with those of domestic cattle. Based on the minimum spanning tree, B. abortus isolates from wildlife were closely related to or had originated from livestock. Control measures are necessary to be able to block the transmission of Brucella between domestic and wild animals, and continuous monitoring of wildlife will be necessary to eradicate brucellosis in South Korea.