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	3:	Evaluation of diagnostic potential of rOmp28 protein of Brucella melitensis for serodiagnosis of ovine and caprine brucellosis
Sapana Tiwaria, Ashu Kumara, Duraipandian Thavaselvama*, Suresh Namdeo Suryawanshib, Archana Prakasha, Anita Baruaa, Sonia Aroraa, and Kannusamy Sathyaseelana
aMicrobiology Division, Defence Research and Development Establishment, Jhansi Road, Gwalior - 474 002, Madhya Pradesh, India.
bAnimal Disease Monitoring and Surveillance (ADMAS) Collaborating Unit, Western Regional Disease Diagnostic Laboratory (WRDDL), Pune - 411007, Maharashtra, India.


*Corresponding Author: 
Duraipandian Thavaselvam
Email: HYPERLINK "mailto:dtselvam@rediffmail.com"dtselvam@rediffmail.com HYPERLINK "mailto:dtselvam@drde.drdo.in"dtselvam@drde.drdo.in         
Fax - 91 751 2341148
Running Title: Diagnostic potential of rOmp28 for ovine and caprine brucellosis


Abstract 
Brucella melitensis is the main causative agent of brucellosis in small ruminants and the diagnosis of Brucella infection can be made by assessing specific cell-mediated or serological responses to Brucella antigens. The present study was designed to evaluate the diagnostic potential of recombinant Omp28 (rOmp28) antigen of B. melitensis for ovine and caprine brucellosis. The omp28 gene was cloned and expressed in pQE30UA bacterial expression system and purified with Ni-NTA affinity chromatography. This purified rOmp28 was used as antigen in indirect plate ELISA and a total of 163 samples (n=79 samples of ovine and n=84 samples of caprine) were tested and compared with the native antigens [cell envelope antigen (CE) and whole cell sonicated antigen (SA)]. The results were also compared with the conventional Standard Tube Agglutination Test (STAT) and overall sensitivity and specificity of rOmp28 were determined and compared with native antigens and conventional STAT. The rOmp28 antigen showed promising results with sensitivity and specificity 71.4%, 97.7% for ovine samples and 74%, 87.8% for caprine samples as compared with CE antigen [(40%, 75%) and (44%, 67.6%)] and SA antigen [(37.1%, 84%) and (42%, 70.5%)] for ovine and caprine samples respectively. This study demonstrated that rOmp28 protein can be a used as a good candidate antigen for serodiagnosis of ovine and caprine brucellosis and also have the immense potential for further development of rapid field-adaptable diagnostic assay for screening and preliminary diagnosis of ovine and caprine brucellosis.  
Keywords: Brucella melitensis, Indirect ELISA, ovine and caprine brucellosis, rOmp28.

Introduction
The ovine and caprine brucellosis is caused by gram negative, coccobacilli, intracellular, facultative bacteria Brucella melitensis. The disease is characterized by severe economic loss caused by abortion in these small ruminants. This organism contains biovars 1, 2 and 3 and all three biovars cause disease in ovine and caprine. B. melitensis infection in sheep is endemic in Mediterranean region, especially along its northern and eastern shores, stretching through Central Asia, Southern Arabian Peninsula, Eastern Mongolia, parts of Latin America, especially Mexico, Peru and Northern Argentina and also in Africa and India. In India, the seroprevalence of brucellosis in small ruminants have been evaluated throughout the country [1, 2]. The modes of infection are direct or indirect; animals become infected directly by infected aerosols or by uptake of infected material including manure. Another mode of infection is grazing pastures where infected animals pass on the infection to healthy animals through contact. The disease is characterized with abortion, retained placenta, orchitis, epididymitis and rarely arthritis. The organism is excreted in uterine discharges, semen and in milk, shedding of bacterium in semen and milk can be for longer duration and possibly lifelong. In goats, the infection varies in duration from very short period and can be rapidly eliminated in vaccinated animals.  In non-vaccinated animals the excretion of the organism in the milk may continue for two or more lactations. The situation in sheep is very different as they are more resistant to reinfection, though this varies with the susceptibility of the breed [3]. Werschilova and Striedter [4] have demonstrated a strong resistance to re-infection, even during pregnancy up to 8 to 9 months, after which it began to decline and subsequent resistance was demonstrated even after two years. Diagnosis of the disease depends on the isolation of bacteria from aborted material or udder secretions. Presumptive diagnosis of Brucella infection can be made by assessing serological responses to Brucella antigens to screen flocks. Standard Tube Agglutination Test (STAT), Rose Bengal Plate Agglutination test (RBPT), Buffered Plate Agglutination test (BPA), Complement Fixation test (CFT), and more recently Enzyme Linked Immuno-sorbent Assay (ELISA) have been used for the serological diagnosis of brucellosis in sheep and goats. A combination of tests shows a degree of sensitivity and specificity which appears sufficient to detect infected animals. The removal of those infected animals can contribute to disease control when vaccination is avoided. The serological tests based on whole cell extract or LPS are not completely specific and cannot always distinguish reactions due to B. melitensis from cross-reactions to other bacteria, particularly Yersinia enterocolitica O: 9 [5]. To overcome these problems as well as to the sensitivity and specificity of the test system, recombinant proteins are used as antigen in ELISA. The rOmp28 of Brucella melitensis is one of the immunodominant antigens and its diagnostic potential for screening of human brucellosis was established earlier in our previous study [6]. In the present study we have purified the recombinant protein (rOmp28) of B. melitensis and evaluated its diagnostic potential for the serodiagnosis of ovine and caprine brucellosis by indirect plate-ELISA.



Materials and Methods
Serum samples
The sera samples of ovine (n= 79) and caprine (n= 84) brucellosis tested in the study were obtained from Animal Disease Monitoring and Surveillance (ADMAS) Collaborating Unit, Western Regional Disease Diagnostic Laboratory (WRDDL), Pune, Maharashtra, India. Samples collected from Nagpur and Nasik region of Maharashtra State, India and were stored at -20�C till use in STAT and indirect ELISA.
Preparation of recombinant Omp28 antigen
The rOmp28 protein of B. melitensis used as antigen in indirect plate ELISA assay was prepared as described in our previous study [7]. The clone of omp28 gene was inoculated in 10 ml LB media containing kanamycin (25 �g/ml) to prepare starter culture for expression and purification of rOmp28 antigen. This overnight grown culture was used for inoculation in 250 ml media with antibiotic in shake flask and incubated at 37�C until the OD of culture reached to 0.5 for induction. After induction with 1 mM IPTG and incubation of 5 hrs, the cells were harvested and used for the purification of rOmp28 by Ni-NTA affinity chromatography protocol (Qiagen, Germany) under denaturing conditions. The purified protein was dialyzed against 1X PBS (pH 7.2) with 3 changes and stored at -20�C in small fractions. The concentration of the protein was estimated by Lowry�s method and used as antigen. 


Preparation of native antigens
Whole cell sonicated antigen (SA)
Overnight grown culture of B. melitensis 16M was inactivated with 1% formaldehyde for 1 hr and then harvested by centrifugation at 8000 rpm for 15 min at 4�C. The inactivated preparation was checked for viability by spreading on Brucella selective agar.  Cell pellet was washed thrice with 1X PBS and resuspended in PBS for sonication. Cell suspension was sonicated for 3 cycles of 5 min each at 40 W amplitude and 8 sec pulse. The sonicated sample was then centrifuged at 10,000 rpm for 10 min and supernatant was used as SA antigen.
Cell envelope antigen (CE)
The CE antigen was prepared according to the previously described protocol [8] with modifications. The inactivated 100 ml of bacterial culture was harvested by centrifugation at 8000 rpm for 15 min at 4�C and pellet was washed twice with 1X PBS and resuspended in 100 ml of buffer 1 (15 mM Tris-HCl pH 8.0, 0.45 M sucrose, 8 mM EDTA and 0.4 mg/ml lysozyme) and incubated at 47�C for 15 min. The sample was centrifuged and pellet was resuspended in 10 ml of buffer 2 (50 mM Tris-HCl pH 7.6, 5 mM MgCl2, 2 mM PMSF). The suspension was sonicated for 3 cycles of 5 min each at 40 W amplitude and 8 sec pulse and centrifuged at 6000 rpm for 15 min to remove unbroken cells. The supernatant was subjected to ultracentrifugation at 43,500 rpm for 90 min and pellet was finally resuspended in 1 ml of buffer 3 (50 mM Tris-HCl pH 7.6, 2 mM PMSF) and stored at -20�C. The concentration of both the native antigens were estimated by Lowry�s method and used for ELISA.  
Indirect plate-ELISA
An indirect plate-ELISA was standardized for screening the sera samples using purified rOmp28 protein. The protein was diluted to final concentration of 25 �g/ml in 0.05 M carbonate buffer and 100 �l of diluted antigen was coated per well in ELISA plates (Nunc, Denmark). Plates were incubated at 37�C for 1 hr and after incubation, washed thrice with 1X phosphate buffer having 0.05% Tween-20 (PBS-T). Plates were then blocked with 200 �l of 1% BSA at 37�C for 1 hr and after washing with PBS-T, serum samples were added to individual wells at 1:100 dilutions in sterile 1X phosphate buffer and incubated for 1 hr at 37�C. Again the plates were washed with PBS-T and incubated with 100 �l of respective conjugate at 1:1000 dilutions in 1X PBS for 1 hr at 37�C. Polyclonal anti sheep-HRP conjugate (Dako, Denmark) was used for sera samples of sheep whereas polyclonal anti goat-HRP conjugate was used with goat samples. After washing with PBS-T, reaction was developed by the addition of 100 �l of developing solution consisting of o-phenylenediamine and H2O2, plate was incubated for 5-10 min in dark for colour development and reaction was stopped by 10 �l of 1 N H2SO4 per well. The absorbance was read at 495 nm in an ELISA reader. A culture confirmed positive and negative control was included in each run to ensure the accuracy of the test. ELISA was also performed following the same procedure using sonicated antigen and cell envelop antigen for comparison.
Standard Tube Agglutination Test (STAT)
All 163 sera samples of brucellosis were screened with STAT, the conventional serological agglutination assay primarily used for the diagnosis of brucellosis. To detect the antibodies against Brucella in serum samples, plain antigen of B. abortus S99 was used as per the method of Alton et al [9]. Two fold serial dilutions (1:20 to 1:640) of the sera were prepared in phenol saline and equal quantity (0.5 ml) of antigen was added to each tube. All the tubes were incubated at 37�C for 24 hr. The results were compared with the antigen control tube showing 50% agglutination. A titer of 1:40 or above was considered as positive. Agglutination reaction was also performed with culture confirmed positive and negative sample in every run.
Comparison of ELISA with Conventional agglutination test (STAT)
Results of ELISA using recombinant as well as native antigens were compared with STAT considering it as gold standard and sensitivity and specificity of ELISA was calculated as per the following formulae; Sensitivity = [true positives/ (true positives + false negatives)] X100,  Specificity = [true negatives/ (true negative + false positives)] X100, Positive Predictive Value= [true positive/ (true positive + false positive)] X100, Negative Predictive Value = [true negative/(true negative + false negative)] X100, Correlation= [(true positive + true negative)/ total number of samples] X100.  A true positive was defined as a sample positive by the agglutination test as well as by the ELISA and a sample was consider true negative if it was found negative by both the tests evaluated. False negative sample was classified as it was positive by agglutination tests but negative in ELISA and false positive sample was that which was negative by agglutination tests but positive by ELISA.  Results of ELISA using rOmp28 antigen were also compared with CE-ELISA and SA-ELISA to evaluate the diagnostic potential of recombinant antigen over the native antigens.
Results 
Preparation of recombinant Omp28 and native antigens
The rOmp28 protein was purified from shake flask culture by Ni-NTA affinity chromatography under denaturing conditions and different fractions of purification were analyzed in SDS-PAGE as described earlier [6, 7].  The band of 30 kDa in size for rOmp28 protein was observed in gel the yield of purified protein was estimated at 128 mg/L of bacterial culture.  The concentration of sonicated and cell envelop antigen was estimated as 160 mg/L and 58 mg/L of bacterial culture respectively. The antigens were stored at -20�C in small aliquots for their use in plate-ELISA.
Indirect plate-ELISA with clinical samples of ovine and caprine brucellosis
All the 163 samples were tested by indirect plate-ELISA using recombinant (rOmp28) as well as native (CE and SA) antigens and a cutoff OD value of 0.22, 0.45, 0.46 for ovine samples (Fig 1) and 0.25, 0.5, 0.24 for caprine samples (Fig 2) was determined for rOmp28-ELISA, CE-ELISA and SA-ELISA respectively. Out of 79 ovine samples tested, 26 (32.9%) were found positive by rOmp28-ELISA, whereas 25 (31.6%) and 20 (25.3%) samples were found positive by CE-ELISA and SA-ELISA. A total of 53, 54 and 59 samples were found negative by rOmp28-ELISA, CE-ELISA and SA-ELISA respectively. In case of caprine brucellosis, out of 84 samples tested 42 (50%), 33 (39.3%) and 31 (36.9%) were found positive whereas 42, 51 and 53 samples were found negative by rOmp28-ELISA, CE-ELISA and SA-ELISA respectively. 

Indirect plate-ELISA vs standard tube agglutination test
Samples were screened with conventional tube agglutination method and out of 79 ovine samples tested by STAT, 35 (44.3%) were found positive whereas 44 samples were negative, in case of 84 caprine samples, 50 (59.5%) were positive and 34 samples were found negative. It was noted that 10 sheep and 13 goat samples that were found positive by STAT were detected negative by rOmp28-ELISA. Similarly, 21 sheep and 28 goat samples in case of CE antigen and 22 sheep and 29 goat samples in case of SA antigen were found positive by STAT but negative by respective ELISA. When the sensitivity of all the three ELISAs (rOmp28, CE and SA) were compared with STAT considering it as gold standard test, it was found 71.42%, 40%, 37.14% for ovine and 74%, 44%, 42% for caprine by rOmp28, CE and SA ELISA respectively. The specificity of these three ELISA assays were found as 97.72%, 75%, 84.09% for ovine and 87.8%, 67.64%, 70.58% for caprine respectively (Table 1). Positive and negative predictive values of the ELISA were also determined and only rOmp28-ELISA showed maximum positive predictive value of 96.15% and negative predictive value of 81.13%. The rOmp28-ELISA also showed highest correlation of 79.43% with STAT when compared with ELISA using native antigens (Table 1). The rOmp28 ELISA correlated well in all the statistical parameters with that of STAT with high sensitivity, specificity, PPV, NPV and correlation.
Comparison of rOmp28 antigen with native antigens in indirect-ELISA
The cut off OD was calculated earlier with a set of calibrator sera samples for all the three antigens and was fixed as 0.22 for rOmp28 antigen, 0.45 for cell envelope antigen and 0.46 for whole cell sonicated antigen for ovine and 0.25, 0.5 and 0.24 respectively for caprine samples. In the indirect ELISA using rOmp28 antigen, 54 negative samples had the OD values ranging from 0.026 to 0.21 with a mean of 0.12�0.05, whereas in the positive samples the OD ranged from 0.22 to 0.584 with a mean of 0.33�0.09. 
Discussion
Brucellosis remains a major threat from the zoonotic as well as economic point of view in small ruminants in most countries of the Mediterranean basin, Middle East and Central Asia [10] and infection in sheep and goat occurs naturally with B. melitensis. Symptoms of the disease mimic other infectious diseases and making diagnosis of the disease challenging [11]. Though the diagnosis is generally based on serological testing but one of the most controversial points concerning serological diagnosis of B. melitensis infection in small ruminants is related to which Brucella species and biovars are used in production of antigens. The RBPT and CFT are the most widely used tests for serological diagnosis of sheep and goat brucellosis and these are also official tests for international trade. However, other studies demonstrate the value of BPA, SAT, skin delayed-type hypersensitivity (SDTH) [12, 13] and more recently ELISA [14, 15, 16] assays for the detection of brucellosis in sheep. Although the currently in use serological tests effectively detects brucellosis on a flock basis but still detection of the disease with these tests in areas with low prevalence is problematic because serum titers of some infected animals sharply declines after infection or because infected sheep do not react in the commonly used serological tests [17]. Isolation of Brucella could be a gold standard as it unequivocally establishes the cause of infection. However, bacteriological examination cannot always be relied on to prove the presence or absence of infection in individual animals [9, 18] as facilities for isolation is not commonly available in endemic regions and serological tests are usually accepted for diagnosis of brucellosis.
Encouraging results have been obtained in sheep and goat samples with iELISA or at a lesser degree with iELISA using various antigens, but generally with a high content of S-LPS. However, antibodies against LPS are also induced in animals vaccinated with Brucella sp. attenuated strain. Therefore an important goal in brucellosis research is the identification of protein antigens that induce an intense antibody response during infection and that are not essential for the induced protective immunity or for survival of the bacterium. Thus, iELISA using these antigens would provide similar or better sensitivity than RBPT and STAT like classical tests and it has also been reported that latent infections could be detected earlier by ELISA [19]. In this order several studies have been evaluated the use of OMPs, inner cytoplasmic proteins or cytosoluble protein extract (CPE) [15, 20-23] in ELISA for diagnosis of brucellosis in animals. In the present study we have evaluated the diagnostic potential of an outer membrane protein of B. melitensis (rOmp28) in indirect plate-ELISA format for the diagnosis of ovine and caprine brucellosis. Previously BP26, a periplasmic protein have been evaluated for its usefulness as a diagnostic antigen for sheep brucellosis that is caused by B. melitensis or B. ovis [14, 15, 24]. In the study, rOmp28 was prepared as per the standard protocols described earlier and concentration of the protein was found similar to our previous studies [7]. This recombinant antigen was used in indirect plate-ELISA to screen the positive and negative samples of sheep and goat and results of rOmp28-ELISA were compared with ELISA performed with native antigens (CE and SA). All these three ELISA results were also compared with the results of conventional assay (STAT).  When compared with total number of positive and negative samples tested in indirect plate-ELISA as well as with conventional methods, rOmp28 was found to react with maximum positive samples of ovine and caprine, compared to native antigens. The seroprevalence was also determined by indirect ELISA and STAT and variation was observed that may be due to the numbers of false positives and false negatives detected by various tests and similar findings was reported by other workers [25, 26]. The sensitivity and specificity of the rOmp28-ELISA was compared for ovine and caprine brucellosis considering STAT as gold standard test, it was found more sensitive and specific than ELISA using native antigens. Results of specificity of all three ELISA were not consistent with respect to antigen used and found higher than sensitivity when compared with STAT for both ovine and caprine, similar results were obtained by other workers [27, 28]. It was suggested that ELISA could replace not only the currently used confirmatory CFT, but also other routine screening tests, namely RBPT and STAT [29]. The use of ELISA in comparison to RBPT and STAT for assessing the situation of brucellosis in cattle with better results is already advocated [30] because chances of non detection of an infected animal in ELISA are minimum when compared with other tests. These findings endorse the diagnostic potential of recombinant outer membrane protein (rOmp28 antigen) of B. melitensis in an indirect plate-ELISA over the native antigens for serodiagnosis of ovine and caprine brucellosis.
Conclusion
The result of indirect plate-ELISA using rOmp28 antigen was compared with native antigens for diagnosis of brucellosis in ovine and caprine clinical samples. In comparison with conventional STAT assay very high sensitivity and specificity was observed with rOmp28 ELISA. The results conclude the reliability of the rOmp28 antigen indirect ELISA in the serodiagnosis of caprine and ovine brucellosis. Since a single serological test may not be enough in the brucellosis disease diagnosis, rOmp28-ELISA system in conjunction with other serological tests like STAT would be used for screening of large numbers of samples.  
Acknowledgement
Authors are thankful to Director, DRDE Gwalior and Head of Microbiology Division, DRDE, Gwalior for their kind supports and providing facilities for this work. 
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