Recombinant Omp2b antigen-based ELISA is an efficient tool for specific serodiagnosis of animal brucellosis

Abstract

Control of brucellosis as a worldwide zoonotic disease is based on vaccination of animals and diagnosis of infected cases to be eradicated. Accurate and rapid detection of infected animals is of critical importance for preventing the spread of disease. Current detection of brucellosis is based on whole-cell antigens and investigating serum antibodies against Brucella lipopolysaccharide (LPS). The critical disadvantage is misdiagnosis of vaccinated animals as infected ones and also cross-reactions with other Gram-negative bacteria. Recombinant outer membrane protein 2b (Omp2b) of Brucella abortus was evaluated as a novel serodiagnostic target in comparison to conventional tests which are based on LPS. Recombinant Omp2b (rOmp2b) was expressed in Escherichia coli BL21 and purified by Ni²⁺-based chromatography. rOmp2b was evaluated in an indirect enzyme-linked immunosorbent assay (ELISA) system for diagnosis of brucellosis, with sera from Brucella-infected mice along with negative sera and sera from mice which were inoculated with other Gram-negative species for assurance of specificity. Thereafter, cattle sera collected from different regions were assessed along with known negative and known positive serum samples. We found that Omp2b can discriminate between Brucella-infected animals and non-infected ones. Results for assessment of two hundred and fifty cattle sera by Omp2b-based indirect ELISA which were compared to Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) showed that our proposed procedure has the sensitivity of 88.5%, specificity of 100%, and accuracy of 90.8%. We suggest that recombinant Omp2b could be used as a protein antigen for diagnosis of brucellosis in domestic animals and can be evaluated for detection of human brucellosis.

Introduction

Brucellosis is a widespread zoonotic disease while its etiological agent, Brucella, is a Gram-negative, facultative intracellular bacterium. Brucellosis is a worldwide spread zoonosis infecting both animals and humans, especially in developing countries [1, 2]. Brucellosis is usually acquired after consumption of contaminated foods, especially dairy products, nonpasteurized or after occupational exposure to infected animals [3, 4]. The prevalence of the disease is very high in Iran [5], Turkey [6], and India [7, 8]. Vaccination of livestock by conventional attenuated strains, the B. melitensis Rev-1 and B. abortus S19, is the main strategy for controlling the disease in animals but could not protect humans against the disease [9]. Control of the disease is also depending upon efficient methods for screening and detection of brucellosis in livestock and humans [10–15].

The main target for serological diagnosis of Brucella infection is the smooth lipopolysaccharide (S-LPS) from the outer membrane of smooth strains [10, 14, 16–18]. Antibody titers against the O-polysaccharide portion of LPS last for a long time in the serum and there are no efficient method for discriminating antibodies elicited according to vaccination and infection [19]. Cross-reactions to other Gram-negative bacteria such as Yersinia enterocolitica O9, Vibrio, Escherichia, and Salmonella also occur that cause misdiagnosis of brucellosis in both humans and animals [20–23].

Brucellae are having the typical structure of Gram-negative bacteria with an outer membrane including a vast variety of proteins called outer membrane proteins (OMPs) [24]. Serological investigation of antibodies against Brucella outer membrane proteins significantly helped to overcome the false positive results due to cross-reacting antibodies which recognize O-polysaccharide epitopes and also differentiating vaccinated and infected animals; examples of which include Omp25 [25], BP26 [26], Omp28 [27, 28], and Omp31 [29]. Omp2b of Brucella is classified in the group II of OMPs which is known as major outer membrane proteins [24]. Omp2b is a well-known target for molecular detection of brucellosis which has been reported by some researchers [30–34] along with protective immunity assessment [35]. Here, we described the production and purification of B. abortus recombinant Omp2b protein in E. coli host and evaluating this protein as a serodiagnostic tool for detection of brucellosis in cattle sera.

Materials and methods

Bacterial strains and culture conditions

B. abortus S19, B. abortus 544, B. melitensis Rev-1, and B. melitensis (all from the culture collection in the Department of Bacteriology, Tarbiat Modares University) were routinely cultured on Brucella agar and incubated in 37 °C for 72 h. Escherichia coli O:157, Salmonella enterica serovar Enteritidis, and Yersinia enterocolitica O:9 were cultured on BHI agar and incubated in 37 °C for 18–24 h. E. coli BL21 (DE3) as prokaryotic hosts for expression of recombinant protein was cultured using either LB broth or LB agar.

Induction, expression, and purification of recombinant Omp2b

Recombinant pET-28a(+) which was inserted with omp2b gene (pET28-omp2b) between BamHI and HindIII sites was previously prepared in our laboratory. Expression and purification were accomplished as previously reported [36]. Briefly, E. coli BL21(DE3) transformed with pET28-omp2b plasmid was grown in an LB medium supplemented with kanamycin (30 μg/ml) while shaking (250 rpm) at 37 °C until the OD_620nm reached 0.6. Protein expression was induced by adding IPTG to a final concentration of 1 mM; thereafter, the culture was incubated for another 2 h. Induced bacterial cells were harvested and purification of the recombinant Omp2b (rOmp2b) was performed by using Ni-NTA agarose through the hybrid method of denaturing-resolubilization procedure [36].

Mouse model for serum preparation

Female BALB/c mice, aged between 6 and 8 weeks were obtained from the Department of Laboratory Animal Production at Pasteur Institute of Iran. Mice were adopted for 1 week before the experiments. Bacterial suspensions with an OD_{620 nm} of 0.08–0.1 which contained approximately 10⁸ bacterial cells were prepared from fresh cultures of B. abortus S19, B. abortus 544, B. melitensis Rev-1, B. melitensis, Escherichia coli O:157, Salmonella enterica serovar Enteritidis, and Yersinia enterocolitica O:9, separately in phosphate-buffered saline (pH 7.2 ± 0.1). Bacteria were heat-inactivated in a 56 °C water bath for 30 min. Separate groups of 10 mice each were inoculated with 50 μL of heat-inactivated bacterial suspension subcutaneously at days 0, 10, 20, and 30 with no adjuvants added. Serum samples were collected from the mice by retroocular bleeding at day 35. A mock group of mice receiving no antigenic component also was considered the negative control. Animal experiments were carried out under Tarbiat Modares Institutional ethics guidelines on laboratory animals and National ethics guidelines for using laboratory animals [37].

Cattle serum samples

Serum samples from 200 cattle confirmed for brucellosis by both Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) were prepared as positive samples. Serum samples from 50 healthy cattle maintained under strict care which were negative for mentioned serological tests were used as negative sera.

Recombinant Omp2b ELISA

The immunoassay plates (Maxisorp, Nunc, Denmark) were coated with 100 μL of purified recombinant Omp2b protein (5 μg/mL) diluted in 0.1 M bicarbonate buffer (pH 9.0) and incubated at 4 °C overnight [36]. The wells were washed three times with phosphate-buffered saline-Tween20 (PBS-T) and then blocked with 250 μL of 5% BSA for 2 h at 37 °C. Two-fold dilutions of mouse sera started at 1:40 prepared in 0.5-mL microtubes (12 dilutions). Immunoassay plates were charged with 100 μL of sera dilutions and incubated at 37 °C for 1 h. After adequate washing with PBS-T, plates were incubated with 100 μL of HRP-conjugated anti-mouse IgG (1:2000) (Sigma) for 1 h at 37 °C. After washing with PBS-T, 100 μL of substrate solution containing TMB and H₂O₂ was added to each well and plates were incubated in the dark at room temperature. Color development was stopped by adding 50 μL of 2 M H₂SO₄ after 15 min. Absorbance recorded at 490 nm wavelength in an ELISA reader. Cattle sera were assessed at a single dilution of 1:100 and detected by HRP-conjugated anti-cow IgG (1:2000) (Sigma) through the same procedure. The relative sensitivity, specificity, and accuracy of recombinant Omp2b-based indirect ELISA were evaluated in comparison to RBPT and SAT as previously described [28].

Results

Production and purification of the recombinant Omp2b

Transformed E. coli BL21 with pET28-omp2b plasmid was grown in an LB medium and the cells induced with 1.0 mM IPTG. rOmp2b was expressed and purified using a previously described hybrid procedure of denaturing-renaturing procedure (Fig. 1). We obtained 3.1 mg of rOmp2b from 1 L of induced culture.

Fig. 1.

Purified recombinant Omp2b 44 kDa on 12.5% polyacrylamide gel which is stained with coomassie blue G-250

Serodiagnostic evaluation of rOmp2b by indirect ELISA

Indirect ELISA experiment with mouse sera immunized with Brucella and non-Brucella whole-cell antigen showed that rOmp2b can specifically detect Brucella-specific antibodies (Fig. 2). There was no significant cross-reaction between Escherichia coli, Salmonella enterica serovar Enteritidis, and Yersinia enterocolitica O:9 and Brucella (p < 0.001).

Fig. 2.

Titration of serum IgG of mice against rOmp2b antigen. Mice immunized with whole cell of B. melitensis 16M, B. melitensis Rev-1, B. abortus 544, and B. abortus S19 did not show any significant difference in reacting with rOmp2b (p > 0.05). There were no significant reactions between rOmp2b and sera from Escherichia coli O:157, Salmonella enterica serovar Enteritidis, and Yersinia enterocolitica O:9 (p > 0.05). There are significant differences between sera titers from mice which received Brucella antigens and those received non-Brucella antigens (p < 0.05) which confirms that there are no cross-reactions

An indirect ELISA experiment was performed with cattle sera at a fixed dilution of 1:100. Cut-off was determined by assessment of negative cattle serum results as mean OD_490nm value +3 × SD (standard deviation) that was 0.162. Table 1 presents the result for cattle sera which were positive for RBPT and SAT and evaluated by indirect ELISA against the rOmp2b. The sensitivity, specificity, and accuracy of recombinant Omp2b-based ELISA relative to RBPT and SAT are 88.5%, 100%, and 90.8%, respectively.

Table 1.

Serological diagnostic values of rOmp2b-based indirect ELISA in comparison to Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) which are totally referred to as “Sero” here

	Sero (+)	Sero (−)	Total
rOmp2b-ELISA (+)	177	0	177
rOmp2b-ELISA (−)	23	50	73
Total	200	50	250

Sensitivity 88.5%, specificity 100%, accuracy 90.8%

Discussion

Brucellosis is a highly contagious zoonotic infection that affects people worldwide and cause significant economical loses [3, 38, 39]. Brucellae are highly infectious and there is currently no confirmed method of preventing human brucellosis; hence, attention should be directed toward effective control and eradicate of the disease in livestock [3, 40]. Brucellosis, in particular infections with B. abortus, B. melitensis, or B. suis, remains a significant human health threat [3, 38]. Although vaccination is the main control measure, administration of currently available vaccines alone is not sufficient for elimination of brucellosis in any host species [40, 41]. Rapid and accurate identification of infection in livestock is crucial for controlling the disease among both humans and animals. Especially in an endemic area, finding a reliable diagnostic method for brucellosis is still an important challenging problem [42]. The detection of antibodies by serological methods is very useful in the diagnosis of brucellosis in both humans and animals. Serological tests such as RBPT and SAT are routinely used in surveillance and control programs of animal brucellosis, but none of the available tests have been shown to be specific and accurate because of false positivity resulting from cross-reactions with other Gram-negative bacterial pathogens and inability to discriminate between vaccinated and infected animals [10].

In the present work, recombinant Omp2b was successfully produced using pET28a (+)/E. coli BL21 system. This recombinant outer membrane antigen was used to develop an indirect ELISA for detection of brucellosis. There are some reports in which protein antigens of Brucellae were used for the serological diagnosis of the disease and overcoming the cross-reaction problem of LPS-based serodiagnostic methods. Among previously investigated protein antigens for detection of brucellosis by ELISA method are Omp10 [43], Omp19 [43], Omp25 [44], Omp28 [28, 43–46], Omp31 [29, 43–45], BP26 [47–50], CP24 [50, 51], and lumazine synthase [50, 52]. These works confirmed that using protein antigens, especially outer membrane proteins of Brucella cell, is very useful in an efficient and accurate serological diagnosis of the disease.

Here, we used BALB/c mice for investigating the diagnostic ability of rOmp2b and its specificity, as described before [44]. Titration of sera from mice following injection of Brucella whole cell showed that specific antibodies against Omp2b are developed which can be detected by indirect ELISA with a significant affinity to the protein antigen. Although we did not detect a significant difference between sera from mice which were immunized with different strains of Brucella, results confirmed that the affinity of the antigen-antibody is high enough to suggest Omp2b as a serodiagnostic target. Sera from mice which received Escherichia coli O:157, Salmonella enterica serovar Enteritidis, and Yersinia enterocolitica O:9 did not react significantly with rOmp2b in ELISA; approving the specificity of the protein antigen against those bacterial species to which cross-reactions may occur in RBPT or SAT.

Ability of rOmp2b-based indirect ELISA to detect brucellosis in an endpoint method was assessed by a total of 250 cattle sera including 50 healthy and 200 infected samples. Twenty-three sera from those that were positive for RBPT and SAT were negative for rOmp2b-ELISA while none of healthy sera reacted with rOmp2b. These results suggest that rOmp2b is able to specifically detect antibodies elicited upon infection with Brucella, and sera which are falsely positive with LPS-based serological methods may be excluded.

Results showed that indirect ELISA, using recombinant Omp2b protein as the target antigen, yielded high sensitivity and specificity for serological diagnosis of brucellosis by detecting specific antibodies in the sera from infected cattle. The same results also may be achieved by evaluating serum samples from other animals like sheep, goat, and dog along with its possible potential for serological diagnosis of human brucellosis.

Compliance with ethical standards

Animal experiments were carried out under Tarbiat Modares Institutional ethics guidelines on laboratory animals and National ethics guidelines for using laboratory animals.