Brucellosis is the lion's share of infectious disease of animals and it has a particular socio-economic importance for the Republic of Kazakhstan. Sixty percent of epizootic outbreaks of brucellosis identified in the Commonwealth of Independent States (CIS) originated from Kazakhstan in recent years. Definitive diagnosis of brucellosis remains a difficult task. Precisely for this reason, we evaluated a purified recombinant out membrane protein 28 (rOMP28) of Brucella species (Brucella spp.) produced in Escherichia coli (E. coli) as a diagnostic antigen in an Indirect ELISA (I-ELISA) for bovine brucellosis. The gene encoding OMP28 was synthesized using a two-round PCR procedure. In order to produce the rOMP28, the de novo synthesized DNA was cloned into the expression vector pET-22b(+). Then, the rOMP28 was expressed in E. coli system and characterized in the present study. We further estimated the diagnostic potential of purified rOMP28 of Brucella spp. for screening bovine sera. To determine if rOMP28 has a valuable benefit for use in the serodiagnosis of bovine brucellosis, rOMP28-based I-ELISA was performed. Brucella spp. positive (n=62) and Brucella spp. negative (n=28) samples from tube agglutination test (TAT) were positive (n=59) and negative (n=27) by I-ELISA, respectively. These findings show that the rOMP28 of Brucella spp. could be a good candidate for improving serological diagnostic methods for bovine brucellosis.
In this article, we describe an immunochromatographic test system developed for rapid serodiagnostics of cattle brucellosis using two markers: Gold nanoparticles (GNPs) and quantum dots (QDs). The test system was compared with immunochromatographic serodiagnostics systems that use only one marker. The approbation of the test system was conducted on samples of cattle sera with low, but diagnostically significant titers of specific antibodies. We show that when two conjugates are used, the intensity of the detectable signal increases by 2–3 times compared with the test system using the QD conjugate and by more than nine times compared with the system using the GNP conjugate.
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.
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