Brucellosis is an emerging zoonotic infection and is endemic in many parts of the world including India. Early and accurate detection of
Brucella spp. is important for adequate treatment of brucellosis since misdiagnosis can lead to high case fatality rate [
18]. The current gold standard for the laboratory based diagnosis of brucellosis is based on isolation of bacteria from clinical samples which require long cultivation periods, risk of laboratory acquired infection and culture isolation rate is very low in most cases [
19]. Because of these limitations the need of serological and molecular diagnosis is considered as the best alternative for brucellosis diagnosis. Serodiagnosis is given importance in most endemic areas as the test can be performed easily in limited clinical set up with minimum resources in comparison to molecular techniques. The antibody response to
Brucella consists of the early production of IgM and immediate progress to the production of IgG2 and small amount of IgG1 [
20].
Brucella a facultative intracellular pathogen resides and multiplies in macrophages but once it has completed its intracellular cycle it is released from the cells by lytic and non lytic mechanism and new release
Brucellae induces the humoral response of IgG antibodies. Presently the serodiagnosis of brucellosis is based on detection of anti LPS antibodies but it requires growth of bacterial culture in laboratory and associated risk to laboratory persons which are handing the
Brucella species
. The LPS of
Brucella species shows high similarity with other bacterial species like
Y. enterocolitica, E. coli, Salmonella species etc. and thus have a chance of cross reactivity with related bacterial species and hence less specific
. Therefore there is an urgent need to develop the diagnostic system based on recombinant antigens and several reports are available based on Outer membrane proteins (Omps) as the antigen of choice to be used in sero diagnosis [
12].
Omps being the major immunoreactive components in the bacterial cells and hence has received the most attention as antigen candidates for development of new diagnostic assays.
Brucella Omps, have been classified according to their molecular masses; group 2 porins (36-38kda) and group 3 proteins (25-27kda) [
21]. Omps plays an important role in the virulence factors of
Brucella and the diagnostic potential of various Omps of
Brucella species have been studied by various groups. Brucella Omp31 has been identified as an interested candidate for serodiagnosis of
Brucella infections in rams [
22] and in farm goats and sheep having history of abortions induced by
B. melitensis [
23]. Omp28 protein was also found to be highly reactive with brucellosis positive sera of human clinical samples [
24]. In another study, Omp28 also showed reactivity to
Brucella infected cattle, dog, sheep, and goat sera [
25]. Comparative evaluation of the recombinant omp28 and omp31 for diagnosis of human brucellosis by ELISA has also been studied and rOmp28 antigen was found to be more suitable antigen for the clinical diagnosis of brucellosis [
26]. The combined use of the recombinant
B. abortus Omp10, Omp19 and Omp28 proteins for the clinical diagnosis of bovine brucellosis is also reported [
27]. The role and size of each Omps is various but the porin Omps being the bacterial components have important roles in the bacterial survival [
28]. In
Brucella species, Group 2 Omps proteins are identified as porins and among them Omp2a and Omp2b are 39 kDa general diffusion pores with 85% sequence identity. In a study, the immunogenicity of
B. abortus Omp2b has been investigated to discover LPS free protein as new diagnostic candidate. Recombinant Omp2b (rOmp2b) elicited the production of TNF-α and IL-6 from the RAW 264.7 cells after 24 h stimulation. In in-vitro stimulation of rOmp2b, spleen cells from naive mice produced high levels of IFN-γ and low levels of IL-4. In in-vivo rOmp2b immunized mice also produced antigen-specific IgM and IgG antibodies in high titre [
29]. Various studies to investigate gene expression of porins proteins under environmental conditions directly associated with
Brucella pathogenicity have also been completed. Thus rOmps and porin proteins are associated directly with the pathogenicity of
Brucella species and high immunogenicity of rOmp2b is reported. The present study was conducted to evaluate another associated porins protein of
Brucella melitensis 16 M. In order to study Omp2a as candidate antigen for serodiagnosis of
Brucella species, the gene of Omp2a was cloned in pET-SUMO prokaryotic expression vector, and the protein was expressed using BL21 DE3 host cells. The expressed recombinant protein was purified and tested in iELISA format for its reactivity with antisera collected from experimentally infected mice at different time intervals of 15, 30 and 60 days. The antigen showed high reactivity with
B. melitensis 16 M, clinical isolate of
B. melitensis 16 M and
B. abortus S19 sera whereas it showed no reactivity with
Y. enterocolitica. Although
B. abortus S19 vaccine strain for animal but can cause infection in human and reactivity of rOmp2a with antisera of
B. abortus S19 strain showed importance of rOmp2a porin protein in animals acquired infections of human brucellosis. Later, it was tested against well defined clinical sera samples divided into 5 groups. The sensitivity and specificity of the rOmp2a porin iELISA was found to be 93.75 and 95.83% respectively. The correlations of rOmp2a porin iELISA with RBPT, STAT, and RBPT + STAT was also calculated and found to be 94.44 and 96.55% respectively. The high sensitivity and specificity of rOmp2a indicate its potential as an important candidate antigen for serodiagnosis of brucellosis.