Diagnosis of human and canine Brucella canis infection: development and evaluation of indirect enzyme-linked immunosorbent assays using recombinant Brucella proteins.

Brucella canis, a Gram-negative coccobacilli belonging to the genus Brucellae, is a pathogenic bacterium that can produce infections in dogs and humans. Multiple studies have been carried out to develop diagnostic techniques to detect all zoonotic Brucellae. Diagnosis of Brucella canis infection is challenging due to the lack of highly specific and sensitive diagnostic assays. This work was divided in two phases: in the first one, were identified antigenic proteins in B. canis that could potentially be used for serological diagnosis of brucellosis. Human sera positive for canine brucellosis infection was used to recognize immunoreactive proteins that were then identified by performing 2D-GEL and immunoblot assays. These spots were analyzed using MALDI TOF MS and predicted proteins were identified. Of the 35 protein spots analyzed, 14 proteins were identified and subsequently characterized using bioinformatics, two of this were selected for the next phase. In the second phase, we developed and validated an indirect enzyme-linked immunosorbent assays using those recombinant proteins: inosine 5' phosphate dehydrogenase, pyruvate dehydrogenase E1 subunit beta (PdhB) and elongation factor Tu (Tuf). These genes were PCR-amplified from genomic DNA of B. canis strain Oliveri, cloned, and expressed in Escherichia coli. Recombinant proteins were purified by metal affinity chromatography, and used as antigens in indirect ELISA. Serum samples from healthy and B. canis-infected humans and dogs were used to evaluate the performance of indirect ELISAs. Our results suggest that PdhB and Tuf proteins could be used as antigens for serologic detection of B. canis infection in humans, but not in dogs. The use of recombinant antigens in iELISA assays to detect B. canis-specific antibodies in human serum could be a valuable tool to improve diagnosis of human brucellosis caused by B. canis.

Identification of the virB operon genes encoding the type IV secretion system, in Colombian Brucella canis isolates.

Canine brucellosis is a zoonotic disease caused by Brucella canis. The establishment of intracellular replicative niches of B. canis is mediated by proteins secreted by the type IV secretion system, which is encoded by the virB operon. The characterization of such genes has been conducted in other species of the genus, but not in B. canis. We report the design of a multiplex PCR test for the detection of the virB operon genes of B. canis. Primers for each of the 12 genes were designed and evaluated using bioinformatics tools. A multiplex PCR assay was standardized and applied to 36 isolates obtained from infected dogs of Aburrá Valley kennels, as well to the Brucella abortus, Brucella melitensis, Brucella suis and Brucella ovis DNA strains. As a result of the in silico design, a pair of primers for each gene was selected. All species and isolates evaluated showed evidence of the presence of the entire virB operon.