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.     

Evaluation of three different antigens in an indirect enzyme-linked immunoassay for the detection of antibodies against Brucella abortus SRB51 in vaccinated heifers

The live attenuated Brucella abortus SRB51 (SRB51) is a partial O-chain-deprived mutant. The relative lack of the polysaccharide prevents it from inducing antibodies detectable by most of the serological tests used for the diagnosis of bovine brucellosis. The performance of three antigens used in an indirect enzyme-linked immunoassay test for detecting SRB51 antibodies were evaluated. A homogeneous group of twenty-five 10-month-old Hereford heifers was used. The animals were bled on day 0 and then subcutaneously vaccinated with 2 ml of a commercially available SRB51 vaccine (Schering-Plough) containing 1x10(7) to 3.4x10(7) viable cells. Blood samples without anticoagulant for sera obtaining were then collected at days 30, 90, 210 and 360 post-vaccination. To detect the SRB51 antibodies, Brucella ovis hot saline extract, B. ovis RLPS (RLPS), and SRB51-RLPS were used. The buffered antigen plate agglutination test and an indirect enzyme-linked immunoassay (I-ELISA) using the smooth LPS (SLPS) antigen from B. abortus were used as control tests. All the sera samples were negative in the BPA test and in the standard I-ELISA using the SLPS. The SRB51-RLPS and the B. ovis RLPS antigens performed better than the B. ovis hot saline extract antigen.     

An improved immunoblotting technique for the serodiagnosis of Brucella ovis infections

Two antigen preparations, the routinely used Brucella ovis sodium dodecylsulfate-mercapto ethanol extract and a B. ovis Triton X-114-derived detergent-rich phase, were compared under standard conditions for their use in electrophoretic immunoblotting for confirmafory, serological testing for B. ovis infections, by using 88 sera from ram flocks with a history of freedom from B. ovis infections, 80 sera from chronically infected rams, which were shedding B. ovis in their semen at the time of sampling, and 104 sera from a naturally infected ram flock. Blots with the detergent-rich phase as antigen gave better correlation with the serological results from naturally infected rams, exhibited no non-specific staining with sera from the negative group, gave clearer visualisation of specific bands for positive sera, and were equally sensitive when compared to the standard antigen for sera from chronically infected rams.     

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.     

Trials with an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of subclinical genital infections in rams caused by Histophilus ovis and Actinobacillus seminis

The performance of an enzyme-linked immunosorbent assay (ELISA) was evaluated in the serological diagnosis of subclinical genital infection in 6 naturally infected ram flocks and 2 experimentally infected ram hoggets. The test employs lipopolysaccharide (LPS) antigen prepared by autoclaving from Actinobacillus seminis and Histophilus ovis. A total of 193 sheep (118 unmated virgin rams and 75 mature breeding rams) were examined clinically, serologically (by ELISA) and bacteriologically (semen bacteriology) at the same time. Serum samples from all animals were also tested by an ELISA employing LPS antigen prepared from Brucella ovis in the same way. Shedding of A. seminis and H. ovis did not show close correlation with serological positivity (Table 1), as only 9 (15.0%) out of the 60 A. seminis shedders were ELISA seropositive at the same time. As regards H. ovis only 10 (19.2%) out of the 52 H. ovis shedders were ELISA seropositive at the same time. The results indicate that, when used alone, the ELISA employing LPS antigen is unsuitable for diagnosing subclinical genital infection caused by H. ovis and A. seminis in rams. The authors discussed shortly the employing fields of this ELISA test in the diagnostic work.     

The complement fixation test for the diagnosis of Brucella ovis infection in rams--an Animal Health Division Report

Complement fixation tests using three B. ovis antigen preparations in warm fixation tests (WCFT) and cold fixation (CCFT) tests were done on 541 ram sera. Semen samples from the same rams were examined culturally to identify B. ovis excretors. The CCFT, using an antigen prepared by heat extraction of B. ovis cells, had a sensitivity of 97% in 124 rams which were shedding B.ovis. The specificity was 99% in 144 rams from non-infected flocks. Seventy-seven per cent of 156 rams which reacted to this test were shedding B. ovis in their semen. Tests with other antigens were inferior in sensitivity and/or specificity. The WCFT gave lower titres than CCFT. Vaccination caused large numbers of false positive reactions in 4 flocks.     

Evaluation of an enzyme linked-immunosorbent assay for the diagnosis of Brucella ovis infection in rams

A simple enzyme linked immunosorbent assay (ELISA) was developed for the serological diagnosis of Brucella ovis infections in rams. Serums from brucellosis accredited-free flocks and flocks known to be infected with B. ovis were tested and the results correlated with warm complement fixation (CF) test and bacteriological examination of semen. Both the ELISA and the CF test detected 0.5% false positive reactions in rams from clinically negative flocks. However the ELISA detected significantly more positive reactors in infected flocks and the CF test failed to detect some rams excreting B. ovis. The ELISA proved to be a valuable test in eradicating brucellosis from infected flocks.     

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.     

Serology and semen culture for the diagnosis of Brucella ovis infection in chronically infected rams

The serological response to Brucella ovis and the shedding of the organism in semen was followed for a period of 13-14 months in 42 naturally infected rams. Most rams remained chronically infected and excreted the organism in their semen throughout the investigation. B. ovis was isolated from 87.9% of the semen samples from the infected rams. The most common sites from which B. ovis could be isolated at necropsy were the epididymides and accessory sexual glands. In one ram the organism was isolated from lung, spleen, kidney and iliac lymph nodes. Three rams ceased to shed B. ovis in their semen during the course of the investigation. Seventy-five (11%) of 686 sera from infected rams were negative in the complement fixation test (CFT) although 76% and 77% of CFT-negative sera were positive in the gel diffusion precipitin test (GDT) and enzyme labelled immunosorbent assay (ELISA) respectively. The high incidence of CFT-negative infected rams was due to the selection for the investigation of many rams with histories of negative or vacillating CFT titres. Sera from five rams which never shed B. ovis in their semen reacted erratically in the three serological tests. The five rams were from heavily infected flocks and were kept in contact with infected rams throughout the investigation.     

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).     

Immunogenicity of recombinant Omp31 from Brucella melitensis in rams and serum bactericidal activity against B. ovis

Detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli was previously identified as a protective immunogen against B. ovis in mice. In this study, we evaluated the immunogenicity of rOmp31extract in rams. This immunogen was emulsified in an oil adjuvant and administered three times with 4 and 8 weeks intervals. Antibody response was measured in serum by whole B. ovis ELISA. Specific antibodies to purified rOmp31 (pET-Omp31) were detected by Western blotting and indirect ELISA. In addition, isotype specific antibodies were measured in tears. Serum bactericidal activity against B. ovis in the presence of complement was measured in vitro. Cellular immune response was explored by intradermal testing with purified rOmp31. Immunization with rOmp31 extract induced IgG specific antibodies in serum able to bind to whole B. ovis cells. Furthermore, strong inhibition in a competitive ELISA (with an Omp31-specific monoclonal antibody) suggested that a proportion of Omp31-specific antibodies were directed against a loop containing a protective epitope. Serum antibodies killed efficiently B. ovis in vitro in the presence of either guinea pig or ovine serum. Tears had both IgG and IgA antibodies to equivalent titers. Finally, immunized rams showed skin reactivity to Omp31. These data demonstrate that B. melitensis Omp31, a protective antigen identified in the mouse model, induces antibody and cellular immune mechanisms in sheep.     

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.     

Ovine brucellosis in alberta

Two parallel surveys of rams from Alberta sheep flocks were conducted to determine the presence of infection with Brucella ovis. In a retrospective study over a period of 24 months, using complement fixation test, 12 flocks out of 142 tested were considered infected. In another 17-month survey of slaughter rams by serology and culture methods 11 flocks out of 124 were found to be infected. The overall prevalence of ovine brucellosis was 8.6% of the flocks tested which represented 12.5% of the estimated sheep flocks in Alberta. Up to 67% of rams in infected flocks reacted to complement fixation test.

The complement fixation test was evaluated for its efficiency in the diagnosis of ovine brucellosis and compared with a limited number of an enzyme-linked immunosorbent assay (ELISA) results and clinical criteria. The complement fixation test as well as ELISA identified all culture positive rams. Both serological tests appeared satisfactory for the diagnosis of B. ovis epididymitis when the results could be interpreted in the light of flock history and clinical findings.

     

Identification of immunoreactive membrane antigens of Brucella ovis by ELISA profiling

Enzyme-linked immunosorbent assay (ELISA) profiling was used to identify the immunoreactive membrane antigens of Brucella ovis. Immunoreactive membrane antigens obtained after detergent extraction of the bacterial membrane complex (inner and outer membranes) were resolved into five peaks (A, B, B1, C and D) by gel permeation chromatography. Aliquots from each of the chromatographed fractions were coupled to 96-well microtitre plates and immunoreactive fractions identified with sera from two rams. Serum from ram 1 which had been vaccinated with a single injection of formalin-killed B ovis emulsified in incomplete Freund's adjuvant identified A and B as the major immunoreactive peaks. Serum from ram 2, which had been successfully infected with B ovis, reacted mainly against peaks A, B1, C and D. This observation facilitated the use of A, B, B1, C and D peak antigens as test reagents to examine the serological response of 12 other rams exposed to B ovis by vaccination or intraconjunctival or intravenous inoculation. Sera from rams which developed productive infections reacted strongly against peaks A, B1, C and D while vaccinated rams had preferential antibody activity against peaks A and B.     

Temporal ELISA response of rams to Brucella ovis following experimental infection or vaccination

Sera from rams vaccinated with antigens extracted chaotropically from Brucella ovis by potassium thiocyanate treatment were used to optimise a whole-cell, enzyme-linked immunosorbent assay (CELISA) and to monitor the temporal serological response of rams which had been challenged with infected semen by the intranasal or intrapreputial route. Three patterns of CELISA response were detected. Thirteen of 15 rams intranasally challenged did not respond serologically (pattern 1 or nil response). Only one of 15 rams in the intranasal group exhibited a rise and fall response with CELISA (pattern 2), while another showed a rise and surge response (pattern 3). The numbers of rams in the intrapreputial group which displayed a pattern 1 or 2 or 3 response were four, nine and two, respectively. No ram with a pattern 2 response excreted B ovis in the semen or showed any other evidence of infection, whereas rams with a pattern 3 response excreted B ovis in the semen and developed palpable lesions. Intrapreputially challenged rams that were CELISA-positive consistently mounted an antibody response against B ovis about two to four weeks earlier than intranasally challenged rams.     

Evaluation of electrophoretic immunoblotting for Brucella ovis infection in deer using ram and deer serum

AIMS: Recently the first case of natural infection of deer with Brucella ovis was discovered. The aim of this study was to develop and evaluate an electrophoretic immunoblotting method for testing deer serum for specific B. ovis antibodies. METHODS: An existing immunoblotting method for sheep serum was altered by using a recombinant protein G-alkaline phosphatase conjugate and Tris-buffered saline containing 3% non-fat dry milk powder for the blocking step and the serum and conjugate dilutions. The method was evaluated using 106 sheep sera from B. ovis - negative, accredited flocks, 69 sera from chronically infected rams shedding B. ovis in their semen, 110 sera from a B. ovis-infected flock, 18 sera from stags from which B. ovis was isolated, and 48 sera from deer flocks free from B. ovis infections. The immunoblotting method was applied to another 85 deer sera. RESULTS: The sensitivity of the new immunoblotting method was 98.6% for sheep and 94.4% for deer, and the specificity 99.1% for sheep and 100% for deer. Sixty-nine out of 97 deer sera, originating from the property from which the first B. ovis deer case had been reported, tested positive or suspicious in the complement fixation test. Of these, 53 sera exhibited staining patterns in blots typical for B. ovis infections and also one serum which was negative in the CFT. Only six out of 1498 deer sera. from throughout New Zealand had positive or suspicious reactions in the B. ovis complement fixation test. Of these, one exhibited a staining pattern in the blot suggestive of a B. ovis infection, while four showed patterns of suspicious reactions. CONCLUSION: The new immunoblotting technique is useful as a confirmatory serological test method for B. ovis infections in deer.     

Observations on the eradication of Brucella ovis infection from a ram flock

The measures taken to eradicate Brucella ovis infection from a naturally infected flock of 64 rams are described. Lesions of epididymitis were detected in 18 rams, all of which gave either positive or suspicious reactions in the complement fixation test. A further 20 rams gave serological reactions in the complement fixation test. Subsequently, semen was collected from 14 of these 20 rams and B. ovis was cultured from the semen of all 14 rams. Serum samples from two rams failed to react in the complement fixation test. However, they were identified as infected with the aid of an enzyme-linked immunosorbent assay and the subsequent culture of semen samples. It is suggested that, when eradicating B. ovis infection from ram flocks, the enzyme-linked immunosorbent assay be used in addition to both the complement fixation test and the physical examination. Using a combination of tests as described can increase the likehood of an earlier eradication of B. ovis infection.     

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.     

Epidemiological Observations in a Corriedale Flock affected by Brucella ovis

Brucellosis in sheep, caused by Brucella ovis, is primarily a chronic infectious disease of rams with epididymitis as its most characteristic lesion. Six hundred rams from an infected farm were clinically and serologically examined once a year, over a 3-year period. An increase from 2.1% to 6.3% in the prevalence of animals serologically positive to B. ovis occurred over the 3 years. However, the prevalence of rams with lesions in the reproductive tract declined from 14.2% to 6.5% in the third year following one year of strict culling of clinically affected and rams that were serologically positive for B. ovis. Clinical lesions found in the 179 affected rams fell into two main categories: rams with epididymitis and rams with affected lymph nodes. These results suggest that the prevalence of the disease relates mainly to the sexual activity of the animal and not to age in itself. A single cull based on the results of clinical examination and serological test results was unable to decrease the prevalence of B. ovis in an extensive Corriedale sheep flock.     

Examination of rams culled during an ovine brucellosis accredited free flock scheme

During the early stages of an Ovine Brucellosis Accredited Free Flock Scheme in New South Wales 62 rams were examined to determine the status of their flocks of origin with regard to infection with Brucella ovis. Forty rams were selected because they were either single reactors to low titre or one of a small number of reactors in a B. ovis complement fixation test of the whole ram flock. Twenty two rams were selected because they had palpable abnormalities within the scrotum but were negative serologically. After serological, pathological, bacteriological and histological examinations they were classified in the ensuing categories: 7 positive, 7 inconclusive, 26 false positive and 22 with other lesions. The usefulness of this classification, particularly within the accreditation scheme is discussed.