Single-step purification and evaluation of recombinant BP26 protein for serological diagnosis of Brucella ovis infection in rams

To investigate the value of the BP26 protein in the serological diagnosis of ovine brucellosis caused by Brucella ovis, recombinant BP26 protein was produced in Echerichia coli and purified for use in an indirect enzyme-linked immunosorbent assay (I-ELISA). The majority of the recombinant protein was recovered from the supernatant of sonicated recombinant E. coli cells in a soluble form. This facilitated the purification of the recombinant BP26 protein which was achieved by using ion-exchange chromatography. After one step of purification, the purity of the recombinant BP26 protein was analyzed by using SDS-PAGE, Coomassie blue staining, and Western blot with a monoclonal antibody (MAb) directed against the BP26 protein. The degree of purity appeared satisfactory so that it could be directly used in I-ELISA. Although the recombinant BP26-ELISA appeared less useful than I-ELISA using the B. ovis hot saline (HS) extract as antigen, the high number of sera from B. ovis infected rams found positive (90%) in the recombinant BP26-I-ELISA indicated that the BP26 protein may be an additional suitable antigen for serological diagnosis of B. ovis infection in rams.     

Comparison of seroreactivity of rams with brucellosis in a complement fixation test, whole cell ELISA and by immunoblotting

In rams with ovine brucellosis, a high degree of serological correlation exists between the complement fixation (CF) test which utilises antigen extracted from bacteria with hot saline, and the ELISA reactivity using methanol-fixed Brucella ovis as the assay reagent. Since the whole cell ELISA (CELISA) detects mainly antibodies against surface antigens of B. ovis, it was concluded that the similar findings of the two serological tests is due in part to the presence of membrane antigens in the CF test antigen following hot saline extraction of intact bacteria. Immunoblots with pooled sera representing different CF titres confirmed that the major immunoreactive antigens of B. ovis were located in four zones: alpha, beta, gamma 1 and 2 with corresponding apparent molecular masses of 55 and 60 kDa; 27 and 29 kDa; 18.5-20 kDa and 17-18 kDa, respectively. These zones of reactivity were consistently present in immunoblots when assayed against different B. ovis isolates even though Coomassie brilliant blue staining of SDS-PAGE gels revealed some differences in polypeptide banding patterns. However, these intensely-stained CBB bands located at 38 and 40 kDa which distinguished three of the seven B. ovis isolates were considerably less reactive in immunoblots compared to polypeptides that were located at positions equivalent to alpha, beta or gamma reactivities. Intensity of immunoblot reactivity against polypeptides located in the alpha, beta and gamma zones intensified with increasing CF titre. Sera with CF titres greater than 32 also tended to react against bands of higher apparent molecular masses located at 65, 70, 73, 78, 80 and 86 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunochemical studies on a Brucella ovis specific protein antigen

A Brucella ovis surface protein antigen (P-II), obtained by gel filtration with Sepharose 4B of a hot saline extract was characterized. The analysis of P-II over gradient sodium dodecylsulfate electrophoresis yielded an 18.5 and a 20 kDa band. In a radioimmunoprecipitation assay using P-II labeled with 125I, the antigen reacted specifically only with sera from rams experimentally infected with a naturally occurring rough strain of B. ovis and did not react with sera from rams experimentally infected with other smooth Brucella strains (B. abortus and B. melitensis).     

Comparison of the enzyme-linked immunosorbent assay and complement fixation test for detecting Brucella ovis antibodies in sheep

A solid-phase, indirect, enzyme-linked immunosorbent assay (ELISA) was compared with the microtitre complement fixation test for detecting Brucella ovis antibodies in 220 ram sera. The ELISA was more sensitive than the complement fixation test; it demonstrated antibodies in 11 sera from known infected or vaccinated rams that were complement fixation test negative. No false positives were recorded with the ELISA and, in 36 sera positive to both tests, the ELISA titres were consistently higher than the corresponding complement fixation test titres.     

Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.     

Characterization of Brucella ovis surface antigens

A rough antigen (SRA) extracted from Brucella ovis in hot saline by Myers procedure, showed three precipitation lines when tested in immunodiffusion against sera from experimentally infected rams. The components responsible for the lines could be isolated by ultracentrifugation or gel filtration which gave 3 fractions, named PI, PII and PIII. The lipopolysaccharide (LPS) appeared in the pellet (SRA-pp) after ultracentrifugation as judged by the presence of lipids, sugar composition, 2 keto-2deoxyoctulosonic acid (KDO), and its characteristic immunoelectrophoretic and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns. SRA-pp contained the antigen responsible for one of the immunoprecipitation lines of SRA and the supernatant (SRA-sn) contained only antigens responsible for the other two. Gel filtration of SRA-pp showed the presence of PI, while SRA-sn gave PII with high protein content and PIII with high carbohydrate content. Immunological activity in gel diffusion (GD) of the Fraction PII and PIII was specific for sera of B. ovis infected rams. Sera from rams experimentally infected with smooth strains (Brucella abortus and melitensis), were not able to react with these antigens.     

Comparison of different antigenic preparations for the detection of ovine serum antibodies against Brucella ovis by ELISA

An enzyme-linked immunosorbent assay has been developed for the detection of antibodies against Brucella ovis using serum from control rams (Con-S), naturally infected rams (Inf-S), rams inoculated intravenously with B. ovis (IV-S) and rams vaccinated intramuscularly (IM-S). The serum was titrated by serial double dilutions from 1/25 to 1/25,600 against whole bacteria, B. ovis lipopolysaccharide and a detergent-extracted component of the outer membrane complex of B. ovis as antigens immobilised on microtitre plates. Sheep antibodies bound to antigen were assayed with rabbit anti-sheep gammaglobulin and alkaline phosphatase conjugated protein A. A high level of antibody activity against intact B. ovis cells was detected in Inf-S and IM-S. When lipopolysaccharide was the immobilised antigen, only IM-S yielded significant antibody activity. The component from detergent extracts of the outer membrane complex of B. ovis reacted best with serum (up to 1/6,400) from field-infected rams, while serum from vaccinated and intravenously inoculated rams registered significant titres up to a serum dilution of 1/800 and 1/200 respectively. These results indicate that ELISA is a very sensitive test but its value as a serodiagnostic procedure is dependent upon the choice of antigen used in the assay.     

Use of Brucella canis antigen for detection of ovine serum antibodies against Brucella ovis

Brucella ovis causes a genital disease of sheep manifested by epididymitis in rams and placentitis in ewes producing reduced fertility in the flock. Clinical diagnosis is not sensitive enough and bacteriological testing is not feasible for detection of the disease in large numbers of animals. Indirect methods of serological testing are preferred for routine diagnosis, of which agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA tests are recommended as the most efficient. Since B. ovis shares antigenic components with Brucella canis, it would seem that either strain could be used as antigen with the same results; however, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination tests. We present data on AGID and IELISA tests using B. ovis antigen and rapid screening agglutination test (RSAT), 2-mercapto-ethanol RSAT (2ME-RSAT) and IELISA using B. canis antigen. We tested 225 animals. The cut-off values were adjusted by ROC analysis using 51 negative and 32 positive sera; the IELISA-B. canis cut-off value was 39 (%P) and IELISA-B. ovis, 51 (%P), with 100% sensitivity and specificity. Of the 32 positive sera from the infected flock RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Of the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, IELISA-B. canis and IELISA-B. ovis, 20 positive only with RSAT and 2 positive only by both IELISAs. RSAT is a very sensitive screening test that, because of its simplicity and easy interpretation, following a study in larger sample, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis. The IELISA-B. canis or IELISA-B. ovis could be used as confirmatory tests, since they show equal specificity and sensitivity.     

Comparison of an enzyme-linked immunospecific assay (ELISA) with the cold complement fixation test for the serodiagnosis of Brucella ovis infection

An enzyme-linked immunospecific assay (ELISA) for the serodiagnosis of Brucella ovis infection in sheep is described and compared with the cold complement fixation (CF) test. ELISA was performed in microtiter plates, using horse-radish peroxidase conjugated to anti-normal sheep serum globulins, and hydrogen peroxide plus o-phenylenediamine as substrate. A heated, cell-free B. ovis extract was used as antigen in both tests. ELISA was easier to perform, distinguished better between positive and negative sera, and did not need heat-inactivated sera.     

A competitive enzyme immunoassay for the detection of bovine antibodies to Brucella abortus using monoclonal antibodies

A competitive enzyme immunoassay (EIA) for the detection of circulating bovine antibodies to Brucella abortus has been developed using horseradish peroxidase conjugated monoclonal antibodies (MAb) raised against B. abortus cell surface antigens. Antibodies present in the serum of either vaccinated or infected cattle can apparently displace the conjugated MAb from the lipopolysaccharide antigen (LPS) in a quantitatively different manner allowing an assessment of immune status of the animal. The results from a panel of sera from animals with a known status of vaccination or infection indicated that the test was more selective in the detection and discrimination of infected from uninfected or immunized animals, than conventional complement fixation, agglutination or indirect enzyme immunoassay procedures.     

Comparison of three serological tests for Brucella ovis infection of rams using different antigenic extracts

The sensitivity and specificity of the complement fixation, gel diffusion and ELISA tests for the diagnosis of Brucella ovis infection of rams have been compared using three different antigenic preparations. The antigens obtained by petroleum ether - chloroform - phenol, or cold saline extractions gave poorer diagnostic results than those obtained by hot saline extraction in all the tests. The best sensitivity was obtained with the ELISA (97.6 per cent) followed by the gel diffusion (96.4 per cent) and complement fixation tests (92.7 per cent). The gel diffusion test detected as positive the two rams negative in the ELISA, while the complement fixation test did not improve the sensitivity of the other tests. Under these conditions all the tests were 100 per cent specific when testing sera from rams free of B ovis.     

Comparison of serological tests based on outer membrane or internal antigens for detecting antibodies to Brucella ovis in infected flocks.

The aim of this work was to compare the performance of 6 serological tests using outer or internal antigens from Brucella for the diagnosis of Brucella ovis infection in sheep in an endemic area. Outer membrane antigens included a hot saline extract (HS) and the rough lipopolysaccharide (R-LPS) from B. ovis. Internal antigens were LPS-free total cytosolic proteins (CP) and an 18-kDa cytosolic protein (p18) from Brucella spp. Sera from 200 sheep from naturally infected flocks were assayed by agar gel immunodiffusion test (AGID) and by complement fixation test (CFT), both using HS, and by 4 ELISA using HS, R-LPS, CP, and p18, respectively. The percentage of positive results was 45.5% for ELISA with HS, 42.0% for ELISA with p18, 39.5% for CFT, 33.5% for ELISA with R-LPS, 29.0% for ELISA with CP, and 18.0% for AGID. Taking CFT as the reference test for calculating relative test parameters, the ELISA with HS had the best sensitivity (96.2%), while AGID and the ELISA with R-LPS had the best specificity (96.6%). The ELISA with CP was not more sensitive than the ELISA with p18 (67.1% vs. 79.7%) in spite of the higher number of antigens in CP. The lower relative sensitivity of tests using internal antigens might reflect a lack of antibodies to cytosolic proteins in some infected animals or a shorter persistence of these antibodies relative to antibodies to outer membrane components after recovery from infection.     

Development of a blocking enzyme-linked immunosorbent assay for detection of turkey antibodies to Mycoplasma meleagridis

A blocking enzyme-linked immunosorbent assay (B-ELISA) was developed to detect antibodies to Mycoplasma meleagridis (MM) in turkey sera. This assay was based on two mouse monoclonal antibodies recognising all MM strains tested but none of seven avian mycoplasmal species tested. Furthermore, their binding to the Tween 20 antigen was inhibited by serum from MM-infected birds. The B-ELISA test format was optimized. The cut-off was determined using a set of sera from MM-free turkeys. This B-ELISA was then compared with a commercial indirect ELISA (I-ELISA). Specificities of the two ELISA tests were not significantly different (100 or 99%, respectively). The sensitivity of B-ELISA was significantly higher than the I-ELISA when I-ELISA suspicious results were considered as negative. Testing sera from experimentally MM-infected animals showed that serum plate agglutination (SPA) test detected positive birds before both ELISA methods. Samples were collected in MM-infected commercial flocks and analyzed by SPA, ELISAs, MM-PCR or culture. Results showed that the sensitivity of the B-ELISA appeared superior to the I-ELISA. Moreover, the ability to detect maternal antibodies makes it a useful tool for eradication or control of MM infections.     

Simultaneous screening of bovine sera for antibodies to bluetongue and epizootic hemorrhagic disease of deer viruses by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (I-ELISA) is described for simultaneous screening of bovine sera for detection of antibodies to bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses (V). Optimal dilutions of BTV and EHDV antigens were combined and allowed to absorb on to the wells of microtiter plates. Appropriately diluted (1:100) bovine sera were allowed to incubate and the bound antibodies were detected by a murine monoclonal antibody (MAb) to bovine immunoglobulin (H-Chain) conjugated with horseradish peroxidase. The performance of the combined (C) I-ELISA in detecting antibodies to BTV and EHDV in sequential serum samples from calves experimentally inoculated with BTV, serotype 10, EHDV, serotype 1 (New Jersey) or EHDV serotype 2 (Alberta) was evaluated. Comparable antibody profiles were demonstrable by the CI-ELISA and separate I-ELISAs using either BTV or EHDV antigens. The results suggest that the CI-ELISA offers many advantages over the standard agar gel immunodiffusion (AGID) test and has potential application as a rapid, sensitive, inter-group-specific and inexpensive test for simultaneous screening of bovine sera for antibodies to BTV and/or EHDV.     

Use of enzyme-linked immunosorbent assay for detection of antibodies to Brucella ovis in sheep: field trial

An enzyme-linked immunosorbent assay (ELISA), using an autoclave-extracted soluble antigen, for the detection of serum antibodies to Brucella ovis in sheep was developed. The test seemed to be both sensitive and specific, on the basis of the control groups studied. The antigen showed no deterioration in prepared plates stored at -70 C for up to a year. The ELISA was used in conjunction with palpation of rams for epididymal lesions as a means to detect and control B ovis infection in a naturally infected flock. All rams were evaluated by the ELISA. At the time that the first blood sample was obtained, all positive and suspicious reactors were removed. With subsequent blood sample collections, only the positive reactors were removed. Brucella ovis was not isolated from any rams during the following year, and none of the mature breeding rams developed epididymal lesions.     

A rapid agglutination assay for canine brucellosis using antigen coated beads

Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Evaluation of surface components of Brucella ovis as antigens for the detection of precipitin antibody in serums from artificially exposed rams

Surface components of Brucella ovis obtained by gentle physical shearing were tested as a potentially useful source of reagent for selective serological diagnosis. These antigens were used in a radial immunodiffusion (RID) test against serum from rams which had been inoculated with infective semen containing B. ovis by one of 4 routes namely mating rams with ewes previously inoculated intravaginally with infective semen, or by direct inoculation in the prepuce, rectum or nasal passage. Loosely attached surface antigens in the RID test formed precipitin bands with serums collected from rams 2 and 10 weeks after inoculation. In contrast, a detergent extracted membrane antigen B developed precipitin bands only with serum collected 10 weeks after inoculation from rams confirmed bacteriologically to be infected with B. ovis in the genital tract. The route by which the rams were artificially exposed did not affect the outcome of the RID test using the membrane B antigen. However, all experimentally exposed rams had demonstrable CF titres when a heat extracted antigen was used.     

A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle

Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi.     

Use of the double immuno gel diffusion test and the enzyme-linked immunosorbent assay to distinguish false from true reactors in the complement fixation test for Brucella ovis

A gel diffusion test with sonicated Brucella ovis antigen and an enzyme-linked immunosorbent assay based on heat-extracted antigen were used to distinguish false from true reactions in a complement fixation test based on heat-extracted antigen. Of 142 complement fixing reactors (occurring in supposedly Brucella ovis-free, accredited flocks), the gel diffusion test correctly identified the status of 139 animals as compared to 128 with the enzyme-linked immunosorbent assay. A combination of the two methods resulted in a correct identification of 141 animals. The procedures provide an easy, cheap and quick way to determine the true status of reactors that show up during routine use of the complement fixation test in Brucella ovis re-accreditation procedures.