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.

Prevalence and molecular detection of Anaplasma marginale, Babesia bovis and Babesia bigemina in cattle from Puntarenas Province, Costa Rica.

A study was conducted in 2008 to determine the prevalence of Anaplasma and Babesia infections in cattle in the Puntarenas Province of Costa Rica. Blood samples were taken from a total of 449 cattle during the month of March at 30 farms in the region of Espiritu Santu, Costa Rica. Commercially available enzyme-linked immunosorbent assays (ELISA) were used to determine presence of antibodies to Babesia bigemina and Anaplasma marginale, and real-time PCR was used to determine the presence of DNA from the disease-causing organisms. The ELISA results indicated that 87.5% of the cattle sampled were positive for antibodies to A. marginale, while 59.1% were positive for antibodies to B. bigemina. The real-time PCR results showed that 235 cattle were carrying A. marginale DNA (56.9%), 6 with B. bigemina DNA (1.34%), and 2 with B. bovis DNA (0.45%).

Evaluation of Brucella abortus DNA vaccine by expression of Cu-Zn superoxide dismutase antigen fused to IL-2.

The Cu-Zn superoxide dismutase (SOD) antigen of Brucella abortus was previously identified to be a T cell antigen which induces both proliferation of and gamma interferon (IFN-gamma) secretion by T cells from infected mice. In an earlier study, we demonstrated that intramuscular injection of mice with a plasmid DNA carrying the gene for SOD leads to the development of significant protection against B. abortus challenge. It has been reported that the antigen-specific immune responses generated by a DNA vaccine can be enhanced by co-delivery of certain cytokine genes. In this study, we evaluated the effect of delivering IL-2 on the efficacy of SOD DNA vaccine by generating a plasmid (pSecTag-SOD-IL2) that codes for a secretory fusion protein of SOD and IL-2. Another plasmid (pSecTag-SOD) that codes for only SOD as a secretory protein was used for comparison. BALB/c mice injected intramuscularly with pSecTag-SOD or pSecTag-SOD-IL2, but not the control plasmid pSecTag, developed SOD-specific antibody and T cell immune responses. Upon in vitro stimulation with recombinant SOD (rSOD) antigen, T cells from mice immunized with pSecTag-SOD-IL2, in comparison with those from mice immunized with pSecTag-SOD, exhibited a lower proliferation response but produced significantly higher concentrations of IFN-gamma. Both DNA vaccines, however, induced similar levels of SOD-specific antibodies and cytotoxic T cell response. Although mice immunized with pSecTag-SOD-IL2 showed increased resistance to challenge with B. abortus virulent strain 2308, this increase was not statistically significant from that of pSecTag-SOD vaccinated mice. These results suggest that a SOD DNA vaccine fused to IL2 did not improve protection efficacy.