Background and Aim: Brucellosis remains one of the most common zoonoses. The current anti-brucellosis measures are largely deemed ineffective due to a lack of specificity of conventional serological tests. This study evaluated the use of Brucella outer membrane protein (Omp)19 for serodiagnostic testing. Materials and Methods: The antigenicity of recombinant Brucella Omp19, Omp25, and Omp31 was examined in serum samples from mice and rabbits immunized with Omp19 or Brucella abortus 19 whole cell (WC) and 12 and 152 cows experimentally or naturally infected with brucellosis, respectively. Serum samples were collected from 151 cows that were vaccinated with B. abortus 19 and 12 unvaccinated heifers that were maintained on a brucellosis-free farm. Results: Immunization with Omp19 resulted in antibody production in mice after a single injection without the use of adjuvant. Serum antibodies obtained from rabbits immunized with inactivated B. abortus strain 19 WC targeted Omps by enzyme-linked immunosorbent assay (ELISA) and Western blot. Antibodies targeting Omp19 were identified in all B. abortus strain 544 experimentally infected cows at day 14 post-inoculation (p.i.); Omp25 was detected by ELISA at day 28 p.i., while an ELISA targeting Omp31 was negative for 25% of cows at this time point. Omp19 and Omp25 were readily detected by sera from cows from a new epizootic focus. Antibodies recognizing Omps were also detected in >50% of the animals maintained in a brucellosis-free herd at 10 months after vaccination. Conclusion: Brucella Omp19 in combination with Omp25 and Omp31 may be utilized as target antigens in an ELISA designed for serological testing of unvaccinated cattle.

The aim of this study was to evaluate the serological diagnostic potential of the Brucella recombinant outer membrane (rOMP25, rOMP31) and periplasmic proteins (rBP26, rSOD) in a comparative way using an indirect enzyme-linked immunosorbent assay (i-ELISA). Rabbit and/or mouse antibodies to Brucella whole cell and/or soluble protein preparations recognized all recombinant proteins used, which confirms the expression of target antigens in E. coli in active form. The recombinant proteins showed different antigenicity to antibodies of cattle kept on a brucellosis-affected (endemic) farm and/or a new focus of infection. Thus, the presence of anti-Brucella antibodies was confirmed by i-ELISA/rSOD in 79% of cows from endemic conditions with positive results by conventional serological tests (RBPT and/or CFT). However, antibodies specific to this protein were detected in only 14% of seropositive animals kept in the hotbed of a new brucellosis infection. Moreover, rSOD-specific antibodies were not detected in the sera of vaccinated cattle from a brucellosis-free farm, whereas antibodies to other recombinant proteins were found in 2%-8% of animals. Using recombinant proteins in immunoassays significantly reduced the number of cows positive for brucellosis. Furthermore, there was not a single protein among the rOMPs that would show the total positive results of all proteins used. Thus, the development of reliable ELISA tests for the diagnosis of brucellosis requires further comprehensive study of the recombinant proteins in order to design a multiprotein antigen that consists of a combination of several proteins with diagnostic potential.

An IgM murine monoclonal antibody (MAb) was obtained against the excretory-secretory antigen (ES-Ag) of in vitro reared protoscoleces of Echinococcus granulosus (Batsch, 1786). Western blotting revealed that the MAb recognised a 20.6 kDa protein of this ES-Ag. The MAb was used in sandwich enzyme-linked immunosorbent assay (s-ELISA) for selective sensitisation of the solid phase with the protoscolex-specific protein from its ES-Ag and somatic antigen (S-Ag) to examine serum samples of 108 cows from a cystic echinococcosis (CE) endemic area for specific antibodies and to compare the results with those from necropsies and an indirect ELISA (i-ELISA). The sensitivity of s-ELISA/ES-Ag, s-ELISA/S-Ag and i-ELISA/S-Ag was 48%, 52% and 62%, respectively. The low sensitivity of the ELISA was probably caused by the fact that 13 cows (62%) were infected with sterile cysts (acephalocysts and/or calcified foci) only. A relatively high specificity (80%) of s-ELISA/ES-Ag was observed in cows with fertile cysts. It also detected antibodies in the serum of two cows that had recovered from the disease according to the necropsy. The i-ELISA/S-Ag gave false results in testing sera from a healthy animal and from a cow with tubercular foci. Further analysis will be necessary to define more precisely the value of this study, because the duration of antibody elimination from the bloodstream of recovered cattle remains unknown. The solution of this problem will increase the specificity of the proposed test in monitoring herbivorous animals for CE.

One of the main links in the system of measures to eliminate brucellosis is the timely and reliable identification of infected animals. In the serodiagnosis of this disease, reactions such as RBPT, CFT (RCFT) and AT are widely used. Recently, various variants of ELISA tests find their application. Both in traditional reactions and in ELISA, lipopolysaccharides of smooth strains of Brucella spp. act as the main antigen, which complicates the differentiating infected from vaccinated animals. In addition, these tests do not always give objective results due to the cross-reactions of Brucella with other gram-negative bacteria. In this regard, the results of studies devoted to the determination of the diagnostic value of the protein components of the pathogen deserve close attention. The diagnostic potential of Brucella recombinant outer membrane proteins (OMP19, OMP25, OMP31) and the periplasmic protein - superoxide dismutase (SOD) in indirect ELISA was studied. The research results showed that cows 10 months after revaccination with  B. abortus 19 in 60% of cases gave positive reactions by RBPT and indirect ELISA based on Brucella OMPs, while antibodies in indirect ELISA/SOD were detected only in 4% of the population. About one third of the suckling calves kept on with their mothers revaccinated against brucellosis had specific antibodies to Brucella OMPs by 6 months of postnatal ontogenesis. The use of individual recombinant proteins in indirect ELISA reduced the sensitivity of the test in serological studies of mother cows and their suckling calves. In serum of seropositive cows from epizootic foci of brucellosis, antibodies to Brucella OMPs as well as SOD were detected in 96.7-100% of cases. Thus, the obtained results provide the basis for further research to determine the serological potential of SOD in the differentiation of Brucella-infected from vaccinated animals.

No abstract