Journal
HELIYON
Volume 6, Issue 7, Pages -Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.heliyon.2020.e04393
Keywords
Biological sciences; Microbiology; Bacteria; Microorganism; Proteins; Proteomics; Veterinary medicine; Health sciences; Infectious disease; Bacteriology; Zoonoses; Immunoproteome: humans; ELISA test; Brucella canis str. Oliveri; Brucellosis
Categories
Funding
- Universidad de Antioquia sustainability strategy 2013-2014
- Comite de Investigaciones Universidad de Antioquia [20127003]
- Colciencias-Colombia [767]
- Vicerrectoria de Investigacion, Universidad de Antioquia [767]
- Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, Indiana, United States
- COLCIENCIAS
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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 50 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.
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