Serological diagnostic potential of recombinant outer membrane protein (Omp31) from Brucella melitensis in goat and sheep brucellosis

Small Ruminant Research 70 (2007) 260–266

Technical note

Serological diagnostic potential of recombinant outer membrane protein (Omp31) from Brucella melitensis in goat and sheep brucellosis V.K. Gupta ∗ , D.K. Verma, S.V. Singh, V.S. Vihan Microbiology Laboratory, Goat Health Division, Central Institute for Research on Goats, Makhdoom, P.O. Farah, Mathura 281122, Uttar Pradesh, India Received 11 July 2005; received in revised form 17 January 2006; accepted 26 January 2006 Available online 2 March 2006

Abstract Outer membrane proteins of Brucella have been classified as group 1 (94 or 88 kDa), group 2 (36–38 kDa), and group 3 (31–34 and 25–27 kDa). Two proteins of 25 and 31 kDa with only 34% of identity are included in group 3 and they are coded for by the omp25 and omp31 genes. Proposed study planned to detect antibodies to Brucella melitensis Omp31 in farm goats having history of B. melitensis induced abortions, in B. melitensis-infected goats and sheep. By enzyme-linked immunosorbent assay (ELISA), using recombinant Omp31 as antigen, of 872 farm goats antibodies to Omp31 were detected in 112 (12.8%) cases. Out of 14 naturally infected goats infected with B. melitensis 12 (85.7%) showed anti Omp31 antibodies. Out of 10 naturally infected sheep with Brucella ovis, antibodies to Omp31 were detected only in 6 (60%) cases and in 18 (81.8%) out of 22 cases infected with B. melitensis. Obtained results were also compared with the rose Bengal plate test (RBPT). In controlled experiments, sensitivity and specificity of recombinant Omp31 (rOmp31) ELISA and RBPT were also evaluated and it was found that former test is 100% specific though RBPT has slightly higher sensitivity. In this study, we found a significant difference between the two groups (B. melitensis and B. ovis infected) in terms of the percentage of positive reactions or signal level by an ELISA. The reactivity of the positive sera against the purified rOmp31 was also tested by Western blotting. Sera from B. melitensis-infected animals showed a strong reactivity in comparison to sera from B. ovis-infected animals. The potential diagnostic usefulness of this antigen in combination with other recombinant proteins from B. melitensis would be of great importance in future in eradication of brucellosis. © 2006 Elsevier B.V. All rights reserved. Keywords: Brucella melitensis; Omp31; Serology; Goats; Sheep

1. Introduction Brucellosis is an infectious disease of worldwide importance in domestic ruminants, and the causative bacteria (Brucella abortus in cattle and Brucella melitensis

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in sheep and goats) are transmitted to humans through contact with infected livestock or by consumption of contaminated dairy products. Because of the economic importance of cattle in developed countries, means for B. abortus diagnosis and prophylaxis have been widely investigated (Alton et al., 1988; Nicoletti, 1990; Plommet and Fensterbank, 1984; Wright and Nielsen, 1990), and several serological tests developed for cattle brucellosis have been found useful for the diagnosis of B. melitensis infection in sheep (Alton, 1990;

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Diaz-Aparicio et al., 1993; Jimenez de Bagues et al., 1992). By contrast, although goats are crucial in the economy of developing countries and B. melitensis is a common cause of human infection, the brucellosis of goats has received comparatively little attention. With respect to serological diagnosis, there have been studies on the use of conventional tests with whole-cell antigens (tube serum agglutination, rose Bengal plate test [RBPT] (Alton, 1990; Falade, 1978; Waghela et al., 1980), and complement fixation test [CFT] (Alton, 1990; Waghela et al., 1980)) and also on agar immunodiffusion tests with uncharacterized trichloroacetic acid or sonic extracts (Waghela et al., 1980; Bell et al., 1976). However, these studies were not performed with sera from goats shown to be actually infected or free of brucellosis, and therefore, the actual value of those tests is unknown. Moreover, tests using fractions known to be antigenically relevant by studies on cattle and sheep brucellosis (Dubray, 1984), including immunoenzymatic tests, have not been investigated. Classical serological techniques rely mainly on the detection of antibodies to lipopolysaccharide (LPS), giving rise to false-positive reactions because of cross-reactivity with LPS from other bacteria. This and other drawbacks of anti-LPS antibodies have generated an increasing interest in the detection of antibodies to alternative antigens, mainly outer membrane proteins (OMPs) and cytoplasmic proteins. Major OMPs from Brucella have been classified in group 2 (Omp2a and Omp2b [36–38 kDa]) and group 3 (Omp25 and Omp31 [25–27 and 31–34 kDa, respectively]) (Cloeckaert et al., 2002). Omp31 was initially cloned from B. melitensis 16 M, and its predicted amino acid sequence revealed a significant homology (34% identity) with Brucella Omp25 (Vizca´ıno et al., 1996). Some observations regarding the Omp31 protein, including its ability to form oligomers resistant to denaturation by sodium dodecyl sulfate (SDS) at low temperatures, suggest that it is a porin (Cloeckaert et al., 2002). Omp31 is expressed in all Brucella species, except B. abortus, which has a 25-kb chromosomal deletion comprising omp31 and other genes (Cherwonogrodzky and Nielsen, 1988). In addition, some differences between Omp31 from B. melitensis and Omp31 from Brucella ovis have been reported. Kittelberger et al. (1998) showed by Western blotting that only 6 of 10 monoclonal antibodies against Omp31 from B. ovis reacted with Omp31 from B. melitensis. These results were later confirmed by Vizca´ıno et al. (1996), who also found that Omp31 from these species differed by seven amino acids. In the present study, we have used purified recombinant Omp31 (rOmp31) from B. melitensis to assess the antibody response to this protein in sera from B. melitensis-

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and B. ovis-infected sheep by an ELISA. Since antiOmp31 antibodies have been sought only in cases of caprine brucellosis, a second goal of the present study was to assess this reactivity in cases of ovine brucellosis. 2. Materials and methods 2.1. Serum samples 2.1.1. Goat sera Eight hundred and seventy-two serum samples from apparently healthy goats of different breeds were collected from an organized farm (where goats were reared under semi-intensive system). In these goats brucellosis is endemic and B. melitensis induced abortions are common. These goats were never vaccinated for brucellosis. Fourteen serum samples from goats naturally infected with B. melitensis were also included. These goats were different from 872 goats described earlier and located at different place. In all cases, the infection was confirmed by isolation of the bacterium from them. All these animals were positive by the rose Bengal plate test and ELISA using sonic extract of B. melitensis as antigen. Serum samples from 12 Brucella free goats located at different place (negative in bacterial isolation as well as serology) were used to calculate the cutoff value of the assay. Blood samples were collected by jugular venipuncture. Serum was stored at −20 ◦ C till further use. 2.1.2. Sheep sera Twelve sera samples from rams naturally infected with B. ovis and 22 sera samples from sheep naturally infected with B. melitensis were also included. These groups of goats and sheep were located at different sites. The infection was confirmed by isolation of the bacterium in all cases. Serum samples from 12 Brucella free sheep located at different place (negative in bacterial isolation as well as serology) were used to calculate the cutoff value of the assay. 2.2. Recombinant Omp31 A 720 bp B. melitensis DNA fragment encoding Omp31 was cloned in pTargeT mammalian expression system vector (Promega, USA) as described (Gupta, NATP-CGP-II-217 Annual Report, 2004). The resultant plasmid (pTargeT–Omp31) contained the Omp31 gene. Competent Escherichia coli JM109 (Promega) was transformed with pTargeT–Omp31. Ampicillin-resistant colonies were grown in Luria–Bertani medium con-

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taining 100 ␮g/ml of ampicillin at 37 ◦ C with agitation (225 rpm). Protein expression was induced by adding 1 mM isopropyl-␤-d-thiogalactopyranoside (IPTG) and incubating transformed cells for 4 h. Bacteria were pelleted by centrifugation (15,000 × g, 20 min, 4 ◦ C) and frozen at −20 ◦ C. Bacterial cells were suspended in a solution consisting of 50 mM Tris, 5 mM EDTA, and 1% Triton X-100 (pH 8.0) (suspension solution) and sonicated for three 1-min cycles at 4 ◦ C. Inclusion bodies were pelleted at 20,000 × g for 30 min at 4 ◦ C and washed twice with suspension solution without Triton X-100. Inclusion bodies were solubilized in a solution containing 50 mM Tris, 5 mM EDTA, and 8 M urea (pH 8.0) at room temperature overnight with agitation. After centrifugation (20,000 × g, 30 min, 4 ◦ C), soluble protein was purified by chromatography through Ni-agarose (Qiagen, United Kingdom). The presence of rOmp31 in eluates was checked by Western blotting with specific MAb A59/10F09/G010 (Cloeckaert et al., 1990). Purity was assessed by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. rOmp31 was adsorbed with Sepharose-polymyxin B to eliminate LPS contamination.

2.5. Western blotting

2.3. Enzyme-linked immunosorbent assay (ELISA) using rOmp31

The sensitivity and specificity of the tests were calculated with respect to the infected and Brucella free groups as described by Jones et al. (1980).

Polystyrene plates (96 wells) were coated with rOmp31 (0.1 ␮g/well) diluted in phosphate-buffered saline (PBS). Unbound sites in the plates were blocked with 200 ␮l of PBS containing 3% bovine serum albumin (BSA) per well. After the wells were washed with PBS containing 0.05% Tween 20 (PBS-T), sera were diluted in PBS-T containing 1% BSA and dispensed to the wells. Specific antibodies were detected with rabbit antigoat HRPO IgG conjugate. The reaction was developed by adding ortho-phenylenediamine (2 ␮g/␮l) in 0.1 M citrate–phosphate buffer containing 0.03% H2 O2 . To establish the cutoff values of the assays, serum samples from non-infected controls were tested under the same conditions. The cutoff value of each enzyme-linked immunosorbent assay system was calculated as the mean specific OD of control sera plus 2 standard deviations (S.D.). 2.4. Rose Bengal plate test Rose Bengal plate test was performed with the commercial cell suspension of B. melitensis (Indian Veterinary Research Institute, Izatnagar, India) standardized according to protocol given by the manufacturer.

To study the influence of denaturation on the antibody recognition of rOmp31, the protein was solubilized in sample buffer and electrophoresed in a 10% polyacrylamide gel in the presence of sodium dodecyl sulfate. The electrophoresed protein was electrotransferred to a nitrocellulose membrane by using the wet procedure. Unbound sites on the membrane were blocked by incubation overnight in Tris-buffered saline (TBS) with 3% BSA. The membrane was washed with TBS containing 0.05% Tween 20 (TBS-T) and cut into strips. Each strip was incubated with caprine/ovine sera (pooled positive sera, mean ODs were 1.143, 0.798, and 0.872 at 492 nm for goat, B. ovis- and B. melitensis-infected sheep, respectively; diluted 1:40) for 3 h at room temperature. After the strips were washed with TBS-T, they were incubated with horseradish peroxidase-conjugated antibodies to goat/sheep immunoglobulins (Sigma) as the case may be for 1 h at room temperature. After the strips were washed again, the reaction was developed with 4-Cl-␣-naphthol and 0.03% H2 O2 . 2.6. Sensitivity and specificity

3. Results and discussion The cutoff value of each enzyme-linked immunosorbent assay system was calculated as the mean specific OD of control sera plus 2 standard deviations. By ELISA, sera from Brucella free goats (n = 12) yielded ODs between 0.092 and 0.272 (mean, 0.158; S.D., 0.038), resulting in a cutoff value of 0.234. Sera from apparently healthy goats (n = 872) from an organized farm having history of abortions yielded ODs between 0.062 and 0.389 (mean, 0.198; S.D., 0.072). Sera from naturally infected goats (n = 14) yielded ODs between 0.111 and 1.212 (mean, 0.589; S.D., 0.203). Of 872 apparently healthy farm goats from a farm having history of abortions, antibodies to Omp31 were detected in 112 (12.8%) cases. Out of 14 naturally infected goats infected with B. melitensis 12 (85.7%) showed anti Omp31 antibodies (Table 1). The result of Omp31 ELISA was compared with RBPT. When these 872 farm goats and 14 naturally infected goats were tested with RBPT, 116 (13.3%) and 13 (92.8%) cases detected positive, respectively. There was total agreement in 98 cases; however, 14 cases were exclusively

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Table 1 Analysis of anti IgG anti-Omp31 antibodies in different groups of goats and sheep S. no.

Status of animals

Total no.

Seropositive

Seronegative

1 2 3 4

Apparently healthy farm goats Naturally infected goats with B. melitensis Naturally infected sheep with B. ovis Naturally infected sheep with B. melitensis

872 14 10 22

112 (12.8%) 12 (85.7%) 6 (60%) 18 (81.8%)

760 (87.2%) 2 (14.3%) 4 (40%) 4 (18.2%)

Table 2 Sensitivity and specificity of rOmp31 ELISA and RBPT in infected (culture positive) and non-infected (culture negative) goats Goats

rOmp31 ELISA

Sensitivity (%)

Naturally infected goats with B. melitensis (infected) Brucella free goats (non-infected)

12/14

85.7

0/12

–

Specificity (%)

100

detected by rOmp31 ELISA but missed by RBPT. On the other end, 18 cases were exclusively detected by RBPT and missed by rOmp31 ELISA. In case of 14 naturally infected goats total agreement was observed in 12 cases; however, 1 case was exclusively detected by RBPT. In case of goats, the rOmp31 ELISA described in the present study appears to be 100% specific, as, in a separate controlled experiment none of the Brucella free goat was detected positive (Table 2), whereas the RBPT found to be 83.4% specific but at the same time sensitivity of RBPT was higher (92%) in comparison to rOmp31 ELISA (85.7%) in same goats (Table 2). Sera from Brucella free sheep (n = 12) yielded ODs between 0.042 and 0.310 (mean, 0.153; S.D., 0.052), resulting in a cutoff value of 0.257. Samples from sheep naturally infected with B. ovis yielded ODs from 0.057 to 0.827 (mean, 0.328; S.D., 0.157), and those from B. melitensis-infected sheep ranged from 0.087 to 0.972 (mean, 0.426; S.D., 0.179). Out of 10 naturally infected sheep with B. ovis, 6 (60%) and 5 (50%) cases were detected positive in rOmp31 ELISA and RBPT, respectively, with total agreement in 5 cases except in 1 case which was exclusively detected by rOmp31 ELISA. The specificity of

RBPT

Sensitivity (%)

13/14

92

02/12

–

Specificity (%)

83.4

rOmp31 ELISA was found to be 100% as none of the Brucella free sheep found positive. The lower sensitivity was observed with RBPT in this group (50%) (Table 3). In case of 22 B. melitensis-infected sheep, 18 (81.8%) and 20 (90.9%) cases were detected positive by rOmp31 ELISA and RBPT, respectively (Table 1), with total agreement in 18 cases except 2 more cases exclusively detected by RBPT and missed by rOmp31 ELISA. The higher sensitivity was observed by the RBPT (90.9%) in this particular group of sheep. However, the 100% specificity was shown by rOmp31 ELISA. Serological tests used in the diagnosis of animal brucellosis can be classified, depending on the antigens used, as conventional tests (i.e., those using suspensions of whole cells as antigens) and tests using antigenic extracts. Conventional tests, RBPT and CFT in particular, are widely used for cattle and sheep brucellosis (Alton et al., 1988), but gel precipitation with selected B. abortus and B. melitensis polysaccharides (AlonsoUrmeneta et al., 1988; Cherwonogrodzky and Nielsen, 1988; Diaz et al., 1979, 1981; Jimenez de Bagues et al., 1992; Jones et al., 1980; Lord and Cherwonogrodzky, 1992; Pinochet et al., 1989) and several ELISAs with S-LPS-rich extracts (Alonso-Urmeneta et al., 1988;

Table 3 Sensitivity and specificity of rOmp31 ELISA and RBPT in infected (culture positive) and non-infected (culture negative) sheep Sheep

rOmp31 ELISA

Sensitivity (%)

Specificity (%)

RBPT

Sensitivity (%)

Specificity (%)

Naturally infected sheep with B. ovis (infected) Naturally infected sheep with B. melitensis (infected) Brucella free sheep (non-infected)

06/10

60

–

05/10

50

–

18/22

81.8

–

20/22

90.9

–

0/12

–

100

01/12

–

91.7

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Jimenez de Bagues et al., 1992; Nielsen et al., 1988) have also been proposed. All the above tests have in common that they detect mostly antibodies to antigenic determinants present in the O chain of the S-LPS. In the present work, the diagnostic potential of Omp31 has been evaluated using sera from goats and sheep with brucellosis proved by bacteriological isolation of bacteria, and the results show that the rOmpp31 ELISA is useful for the diagnosis of B. melitensis infection in goats. However, the RBPT had to be modified by increasing the serum/antigen ratio to reach 100% sensitivity. This confirms (Diaz-Aparicio et al., 1993) that present guidelines for the standardization of the RBPT are not adequate for goat brucellosis. Serological tests that detect antibodies to Brucella proteins have been used in the diagnosis of human brucellosis (Diaz et al., 1976) but seldom in the diagnosis of B. abortus and B. melitensis animal brucellosis. Studies performed by gel precipitation methods have shown that cattle develop antibodies to some Brucella soluble proteins (Dubray, 1984), but no information is available for sheep or goats. To the best of our knowledge, an ELISA using rOmp31 as antigen for the diagnosis of goat brucellosis had not been presented before. The assay described here with rOmp31 is not essentially different from some indirect ELISAs used for animal brucellosis except antigen. When sera from farm goats (872) from area having history of B. melitensis induced abortion were tested, a large discrepancy was found between the rOmp31 ELISA and RBPT. However, this discrepancy was lower when naturally infected goats were tested. The fact evolved in present study that RBPT was more sensitive than rOmp31 ELISA may be explained by a low adherence of the protein antigen (rOmp31) to polystyrene. The discrepancy observed between rOmp31 ELISA and RBPT may be due to the following facts: that RBPT mostly detects the antibodies against the S-LPS; and sera with specific IgM but without significant levels of IgG are positive in RBPT and negative in ELISA using protein antigen (Gupta et al., unpublished data). Presently there is no objective criterion to decide whether cases exclusively detected or missed by either test represent false-positive or negative reactions. This may account for the observed discrepancies in the case of 872 goats which belong to a single farm of unknown infectious status. An improvement of the rOmp31 ELISA would require further research to achieve its binding to the solid phase. According to Vizca´ıno et al. (2001), seven-amino acid difference would explain why some monoclonal antibodies to B. ovis Omp31 and some sera from B. ovis-infected sheep do not react with B. melitensis Omp31. An addi-

Fig. 1. Reactivity by Western blotting of: (Lane 1) standard molecular weight marker; (Lanes 2 and 3) sera from B. ovis-infected sheep; (Lanes 4 and 5) sera from B. melitensis-infected goat; (Lane 6) Mab A59/10F09/G10; (Lane 7) sera from Brucella free goat; (Lane 8) sera from Brucella free sheep.

tional and intriguing finding of their work (Vizca´ıno et al., 2001) was that none of 11 serum samples from B. melitensis-infected sheep reacted with B. melitensis Omp31 by Western blotting, leading the researchers to suggest that this protein does not induce an important humoral immune response in infected sheep. Differences in the reactivity of B. ovis- and B. melitensis-infected sheep to Omp31 have been reported using Western blotting with lysates of transformed E. coli. However, in this study, we found a significant difference between the two groups in terms of the percentage of positive reactions or signal level by an ELISA. The reactivity of the positive pooled sera against the purified rOmp31 was also tested by Western blotting. As shown in Fig. 1, sera from B. melitensis-infected animals showed a strong reactivity in comparison to sera from B. ovis-infected animals. While Omp31 is expressed in all Brucella species, except B. abortus, previous studies using Western blotting have shown a partial or total lack of recognition of B. melitensis Omp31 by sera from B. ovis- or B. melitensisinfected sheep, respectively (Kittelberger et al., 1998; Vizca´ıno et al., 2001). The apparent reduced prevalence of anti-Omp31 antibodies in B. melitensis-infected sheep was attributed to the presence of O polysaccharide chains in B. melitensis LPS, which could lower the immunogenic properties of Omp31 (Vizca´ıno et al., 2001). However, the absence of antibody reactivity against this protein could also be due to the elimination of conformational B-cell epitopes under the denaturing conditions of Western blotting. Antibodies to Omp31 were detected by ELISA in 12.8% cases of farm goats. These results clearly show that goats infected with B. melitensis develop antibodies against the homologue

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Omp31. Here it is important to note that the percentage of B. ovis-infected sheep that reacted with B. melitensis Omp31 by ELISA was lower than that found in previous studies by Western blotting (61% versus 33%) (Vizca´ıno et al., 2001). The detection of antibodies to B. melitensis Omp31 in B. ovis-infected animals clearly shows the presence of shared epitopes between the Omp31 proteins from these species. This finding is also supported by the observation that sera from mice immunized with B. melitensis Omp31 react with B. ovis whole cells by ELISA and with B. ovis Omp31 by Western blotting (Estein et al., 2003). Failure to detect anti-Omp31 antibodies in previous studies may be due to the denaturing conditions of Western blotting, we selected some sera having high reactivity (mean ODs were 1.143, 0.798, and 0.872 at 492 nm for goat, B. ovis-, and B. melitensisinfected sheep, respectively) by ELISA. These sera were tested against the same protein by Western blotting. We found that antibodies to Omp31 were detected in both B. melitensis- and B. ovis-infected animals, unlike the finding of Vizca´ıno et al. (2001). This discrepancy may be due to the usage of a lysate of recombinant E. coli (Vizca´ıno et al., 2001) in contrast to the purified Omp31 used in the present study for the Western blot. Our findings revealed that Omp31 from B. melitensis shares immunogenic epitopes with its homologue from B. ovis and that it can be used to diagnose brucellosis in both B. melitensis-infected goats and B. melitensis- and B. ovisinfected sheep. This is the first study to identify serum reactivity to a particular OMP in goat brucellosis. The findings of this study confirm the diagnostic usefulness of antibodies to Brucella proteins in goat brucellosis, as has been shown for cytosolic components (Baldi et al., 1997). The frequency of occurrence of antibodies to Omp31 in sheep was higher than that reported for other OMPs. In an earlier study using ELISA with recombinant Omp25, 33% of sheep naturally infected with B. melitensis had antibodies to Omp25, and 50% had antibodies to Omp36 or Omp16 (Letesson et al., 1997). The diagnostic value of isolated OMPs has not been assessed in goats. Further studies are required involving large numbers of bacteriologically positive vaccinated goats to reach a definitive conclusion. The potential diagnostic usefulness of this antigen (rOmp31) in combination with other recombinant proteins from B. melitensis would be of greater importance in future in the eradication of brucellosis. Acknowledgments We thank Director, CIRG, Makhdoom, for providing necessary research facility. This work was supported

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