The ELISA for the examination of hare sera for anti-Brucella antibodies

Hare brucellosis is caused primarily by Brucella suis biovar 2. Hares along with wild boars are the natural reservoir of this microorganism. In view of restriction of applicability of traditional serological methods the work aimed to develop the ELISA to examine hare sera for the presence of anti-Brucella antibodies. Lipopolysaccharide (LPS) antigen obtained from the strain S19 of Brucella abortus and the conjugate of antibodies against rabbit immunoglobulin with horseradish peroxidase were used in the test. Hares' sera positive and negative in the CFT were used as controls of the ELISA. The sera collected from 9 hares suspected to be infected with Brucella organisms, positive in CFT (in this number 7 hares revealed clinical symptoms or anathomopathological lesions characteristic of brucellosis), 6 sera from hares showing no symptoms of the disease, negative in CFT and 520 sera from hares monitored for brucellosis were tested. All serum samples from hares suspected for Brucella infection were positive in ELISA and 2 of them were negative in RBPT. Additionally among the samples from hares monitored 12 sera were positive in ELISA and CFT, whereas 9 sera from 12 ones were also positive in the RBPT. The obtained results indicated that the ELISA developed in our laboratory proved to be equivalent in specificity to CFT. In addition, ELISA proved to be more sensitive than RBPT for the diagnosis of Brucella infection in hares.     

Detection of Campylobacter antibodies in sheep sera by a Dot-ELISA using acid extracts from c. fetus ssp. fetus and c. jejuni strains and comparison with a complement fixation test

In this study, a dot-enzyme-linked immunosorbent assay (Dot-ELISA) was evaluated in comparison with a complement fixation test (CFT) for the detection of Campylobacter antibodies in sheep sera. Acid glycine extracts (AGE) of both Campylobacter fetus ssp. fetus and Campylobacter jejuni strains that had been isolated from the gall-bladder of slaughtered sheep was used as antigen in both tests. A total of 153 sheep sera from aborted (74) and slaughtered (79) sheep were examined by both Dot-ELISA and CFT. Twenty-two sera showed anti-complementary activity were not suitable for CFT. Of the 22 sera showing anti-complementary activity, two sera were found to be positive in Dot-ELISA. Eighty-eight (67.2%) of the remaining 131 sera were negative by both Dot-ELISA and CFT using AGE of both Campylobacter strains whereas 43 sera (32.8%) gave different reaction patterns in Dot-ELISA and CFT with the extracts of both Campylobacter strains. Twelve sera were positive by both tests using AGE of C. fetus ssp. fetus but CFT failed to detect antibodies in nine of these sera when AGE of C. jejuni was used. Twelve sera were positive by both tests only when AGE of C. fetus ssp. fetus was used. Eleven sera were positive only by CFT. Seven of these reacted only with the AGE of C. fetus ssp. fetus and four sera were positive by using AGE of both Campylobacter strains. The remaining eight sera were found to be positive only by dot-immunobinding assay either with the AGE of both Campylobacter strains or with the AGE of one of the Campylobacter strains. It is concluded that Dot-ELISA using AGE from C. fetus ssp. fetus could be employed for the detection of Campylobacter antibodies in sheep sera and the additional use of AGE from C. jejuni as antigen appeared not to be profitable for this purpose.     

Comparison of the dot-immunobinding assay with the serum agglutination test, the rose bengal plate test and the milk ring test for the detection of Brucella antibodies in bovine sera and milk.

In this study, Brucella antibodies in bovine sera and milk were detected using the dot-immunobinding assay (DIA), the serum agglutination test (SAT), the Rose Bengal plate test (RBPT) and the milk ring test (MRT). For this purpose, a total of 116 paired blood and milk samples collected at the same time from 56 aborted and from 60 healthy dairy cows was examined. In DIA, a nitrocellulose membrane (NCM) was used as the solid phase. Antigen adsorbed on the NCM was extracted from Brucella abortus S99 by heat treatment. The results obtained by DIA were compared with those of SAT, RBPT and MRT. Of the 116 paired blood and milk samples, 24 were positive and 72 were negative by all tests used. Serum samples of six aborted cows were positive by DIA, SAT and RBPT but the milk samples were negative by DIA and MRT. Serum and milk samples of four aborted cows gave positive reaction only by DIA tests. The remaining six aborted cows were negative only by MRT and two of them were negative by both RBPT and MRT. Four sera of healthy cows were found to be positive only by SAT.     

Comparison of dot enzyme-linked immunosorbent assay (dot-ELISA) with other serological tests for the detection of Brucella antibodies in bovine sera

A dot Enzyme-linked Immunosorbent Assay (dot-ELISA), using whole cell Brucella abortus antigen dotted on the nitrocellulose membrane bound to a plastic strip (dipstick) was employed for the detection of Brucella antibodies in bovine sera. The results were compared with that of serum agglutination (SAT), Rose Bengal plate agglutination (RBPT) and Complement Fixation test (CFT). All the four tests gave negative reaction in 127 sera obtained from a brucellosis free herd. Testing of 549 sera from a chronically infected herd revealed 57 positive and 447 negative animals in all the four assays. Of the remaining 45 sera, 34 were positive in dot-ELISA. Six of these cases were independently detected by dot-ELISA while 28 showed positive reactions in combination with other tests. When serum samples from 158 aborted cases were subjected to dot-ELISA, 79 were found positive. Of these dot-ELISA positive cases, 71 gave positive reaction in SAT, 72 in RBPT and 78 in CFT. B. abortus biotype 3 was isolated from 34 of the 98 aborted fetuses examined.     

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.     

A comparison of three serological tests for the diagnosis of B. ovis infection in rams

The complement fixation test (CFT), the enzyme labelled immunosorbent assay (ELISA) and the gel diffusion precipitin test (GD) were compared, for the diagnosis of Brucella ovis infection in rams. The sensitivities of the tests in 109 rams which were shedding B. ovis in their semen were: CFT 96.3%; ELISA 97.2%; GD 91.7%. The specificities of the tests in 141 rams from non-infected flocks were: CFI 99.3%; ELISA 98.6%; GD 100%. Predictive values of the three tests were measured in 285 rams from infected flocks. Thirty-eight percent of these rams were shedding B. ovis in their semen. Predictive values of positive tests were: CFT 75.5%; ELISA 66.7%; GD 72.5%. Predictive values of negative tests were: CFI 97.1%; ELISA 97.6%; GD 93.8%.     

Comparison of three serological tests in the diagnosis of Brucella infection in unvaccinated cattle in Eritrea

Three serological methods, the Rose-Bengal test (RBT), the complement-fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (I-ELISA) were compared for the detection of Brucella-infected animals in unvaccinated cattle herds in Eritrea. In this study, 71 herds first were classified as positive or negative for Brucella infection on the basis of at least one animal being seropositive by RBT and CFT. All the 159 RBT-positive samples from the 26 seropositive herds and 214 RBT-negative samples randomly selected from the seropositive herds and from the 45 negative herds were tested further by CFT and I-ELISA. Using the ELISA titer as main predictor, and incorporating the RBT results, a logistic model was built to predict the CFT-negative or -positive status of individual sera and to estimate sensitivity and specificity. Whilst the ELISA titers (< or =20) accurately predicted all the negative sera in herds that were also negative by the CFT, the number of seropositive animals was higher by ELISA in herds that had positive animals. Serum samples which give higher degrees of agglutination with the RBT need not be re-tested with CFT; consideration of the seropositive status of a herd should be taken into consideration on defining the cut-off optical density readings for ELISA.     

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 lymphnodes. 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. ovisin 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.     

Brucellosis of camels in Kuwait

This study investigated the presence of Brucella antibodies in serum and milk obtained from camels in Kuwait. Brucella strains were also isolated from the foetus using standard technique (Webridge Lab Techniques). Three serological tests for serum were adopted. These tests were Rose Bengal Plate Test (RBPT), the Serum Tube Agglutination Test (SAT) and the Complement Fixation Test (CFT). The RBPT was used for all sera samples, and both SAT and CET were used for the positive RBPT. Camels that showed a titer of 1:40 in SAT or 1:5 in CFT or greater were considered positive. Thirteen of the samples examined were found positive by CFT (at 1:5); by SAT, they showed a titer of 1:20. One serological test, the Milk Ring Test (MRT), was used for milk. Here 3 and 2 were considered positive reactors but 1+ was considered suspicious. We were unable to isolate the Brucella organism from Sedemine and Cream of milk, but we isolated them from Foetus Brucella abortus and it is confirmed by Webridge Laboratory, U.K. It is Brucella abortus (Biovar 1). The prevalence rate was 14.8% from serum by the CFT and RBPT methods and 10.8% by the SAT method. For milk, the prevalence rate was 8.0%. Two Brucella abortus were isolated from 5 foetuses.     

Evaluation of an enzyme-labeled antiglobulin test for anti-Brucella immunoglobulin G among 3 cattle populations

Antibody to smooth Brucella abortus lipopolysaccharide antigen on the surface of polystyrene tubes was detected with peroxidase-labeled antibody against bovine immunoglobulin G. The enzyme-labeled antiglobulin test (ELAT) activity of samples was expressed in arbitrary units/0.01 ml by reference to a standard curve based on tests of dilutions of a positive serum pool. Reactions greater than 3.0 U/0.01 ml were classified positive because specificity at this level was 99.8% (417/418 samples correctly classified negative) with agglutination test-negative sera from 33 Brucella-free herds. Results of the ELAT were compared with results of agglutination tests and the complement-fixation test (CFT), using 430 sera from cattle in 7 infected herds. Activity of greater than 5.0 ELAT U/0.01 ml was detected in all 54 sera classified as positive (titer greater than 1:10) by the CFT, including 5 sera classified as negative by the tube agglutination test. Sera from 8 nonvaccinated cows in the infected herds reacted only by the ELAT, whereas reactions were obtained with 25 and 5 sera by only agglutination tests and the CFT, respectively. The ELAT and CFT results were in agreement for 25 of 26 sera from agglutination test-reactor cattle in herds of unknown status. Comparisons of milk ring and whey agglutination tests with the whey ELAT on 146 quarter samples from cows in an infected herd revealed no ELAT activity greater than or equal to 1.0 U/0.01 ml in the 73 samples considered negative by the 2 other tests. Samples (n = 47) that contained greater than or equal to 1.0 ELAT U/0.01 ml included all (n = 40) samples with milk ring or whey agglutination titers greater than or equal to 1:16 and greater than or equal to 32, respectively, and 7 samples that gave weaker reactions to the latter tests.     

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.     

Comparison of four serological tests in the diagnosis of caprine brucellosis

Results from four serological tests for diagnosing brucellosis--serum tube agglutination (SAT), agar gel immunodiffusion (AGIT), Rose Bengal plate (RBPT) and complement fixation (CFT)--were compared using sera from goats from farms infected with Brucella melitensis. Ninety-two goats were negative and 29 positive to all four tests. The remaining 85 reacted to one or more tests. The RBPT was the most sensitive test and the AGIT the most specific, when compared with the CFT. The results suggest that the SAT adds little information when used with other tests but that RBPT and AGIT are useful for testing caprine brucellosis where facilities for the CFT are not available.     

Brucellae through the food chain: the role of sheep, goats and springbok (Antidorcus marsupialis) as sources of human infections in Namibia

A confirmed case of human brucellosis motivated an investigation into the potential source of infection in Namibia. Since domestic animals are principal sources of Brucella infection in humans, 1692 serum samples were screened from sheep, goats and cattle from 4 presumably at-risk farms and 900 springbok (Antidorcas marsupialis) serum samples from 29 mixed farming units for Brucella antibodies by the Rose-Bengal test (RBT) and positive cases confirmed by complement fixation test (CFT). To assess the prevalence of human brucellosis, 137 abattoir employees were tested for Brucella antibodies using the standard tube agglutination test (STAT) and by enzyme linked immunosorbent assay (ELISA). Cattle and sheep from all 4 farms were negative by RBT and CFT but 2 of the 4 farms (Ba and C) had 26/42 and 12/285 seropositive goats, respectively. Post mortem examination of seropositive goats revealed no gross pathological lesions typical of brucellosis except enlarged mesenteric and iliac lymph nodes seen in a single buck. Culture for brucellae from organs of seropositive animals was negative. None of the wildlife sera tested positive by either RBT or CFT. Interviews revealed that besides the case that prompted the investigation, a family and another person from other farms with confirmed brucellosis shared a common history of consumption of unpasteurised goat milk, home-made goat cheese and coffee with raw milk and prior contact with goats, suggesting goats as the likely source of infection. All 137 abattoir employees tested negative by STAT, but 3 were positive by ELISA. The 3 abattoir workers were clinically normal and lacked historical connections with clinical cases. Although goats are often associated with B. melitensis, these studies could not explicitly implicate this species owing to cross-reactivity with B. abortus, which can also infect goats. Nevertheless, these data reinforce the need for a better National Control Programme for brucellosis in Namibia.     

Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation

An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.     

Evaluation of a fluorescence polarization assay for the detection of serum antibodies to Brucella abortus in water buffalo (Bubalus bubalis)

The fluorescence polarization assay (FPA) was evaluated for the serological diagnosis of brucellosis in water buffalo (Bubalus bubalis) in southern Italy. This assay uses O-polysaccharide prepared from Brucella abortus lipopolysaccharide conjugated with fluorescein isothiocyanate as a tracer. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. Sera from 890 buffalos from the Campania Region - 526 positive sera and 364 negative sera according to the complement fixation test (CFT) - were evaluated in this study. All samples were tested with the Rose Bengal test (RBT), CFT, and FPA in parallel and in blind fashion. Sensitivities (Sn) were 84.5% and 92.6%, and specificities (Sp) were 93.1% and 91.2% for RBT and FPA, respectively, relative to CFT. Finally, receiver operating characteristic (ROC) analysis suggested a cut-off value of 117 millipolarization (mP) units. On the whole, these results suggested that FPA might replace RBT in the diagnosis of buffalo brucellosis for its better performance relative to CFT, its adjustable cut-off useful in different epidemiological situations, its reliability, ease of performance, and for its potential application in field and high-throughput laboratories.     

Field evaluation of a new commercially available ELISA based on a recombinant antigen for diagnosing Chlamydophila abortus (Chlamydia psittaci serotype 1) infection

A new commercially available ELISA (ELISAr-Chlamydia) for detecting antibodies against Chlamydophila abortus has been evaluated using sheep field serum samples. The ELISA is based on a recombinant antigen which expresses part of a protein from the 80-90kDa family that is specific to C. abortus. Sera (105) from six flocks with confirmed ovine chlamydial abortion (OEA) outbreaks were used in this study, as well as sera (258) from 18 flocks which had suffered no OEA in the last lambing. The ELISAr-Chlamydia was compared with the complement fixation test (CFT) and with an ELISA using purified C. abortus elementary bodies (ELISA-EB), employing as reference technique a comparative microimmunofluorescence test that differentiates C. abortus infection from Chlamydophila pecorum infection. The results showed that the sensitivity of ELISAr-Chlamydia was 90.9% with a specificity of 85.9%, the sensitivity of CFT was 71.0% with a specificity of 83.6%, while the sensitivity of ELISA-EB was 95.2% and the specificity was 54.2%. Furthermore, ELISAr-Chlamydia was the test with fewer false positives resulting from positive reactivity to C. pecorum, although 15% of the sera positive for C. pecorum but negative for C. abortus antibodies reacted positively. This study demonstrated with field material that ELISAr-Chlamydia provides the most balanced results between sensitivity and specificity, especially in flocks with no clinical OEA but reactivity to C. abortus.     

Improved performance of Brucella melitensis native hapten over Brucella abortus OPS tracer on goat antibody detection by the fluorescence polarization assay

The current method for goat brucellosis diagnosis is based on the World Organization for Animal Health (OIE) using the screening card test (CT), with antigen at 8% (CT8) or 3% (CT3) of cell concentrations, and the confirmatory complement fixation test (CFT). However, these tests do not differentiate antibodies induced by vaccination from those derived from field infections by Brucella species or other bacterial agents; in places like Mexico, where the prevalence of brucellosis and the vaccination rates are high, there is a considerable percentage of false positive reactions that causes significant unnecessary slaughter of animals. Furthermore, results of the fluorescence polarization assay (FPA) using the Brucella abortus O-polysaccharide (OPS) tracer in goats are poorer than those with cattle. The present study was undertaken to investigate a tracer prepared from the native hapten (NH) of the Rev. 1 strain of Brucella melitensis to improve FPA performance on goat brucellosis diagnosis. Evaluation of 48 positive samples and 96 negative samples showed that the NH tracer was more accurate (p<0.01) than the OPS tracer (97.2% vs. 93.8% accuracy, respectively). On the diagnostic performance evaluation, the NH tracer performed better (87.5% accuracy, 79.5% sensitivity, 84.3% specificity, and 163.8 performance index) than the OPS tracer (83.5%, 75.9%, 81.0%, and 156.9, respectively) using 1009 positive and 2039 negative Mexican field goat sera samples selected by test series approved by the OIE (card test 3% and CFT). We demonstrated a new application for the NH lipopolysaccharide on detecting antibodies against Brucella using the FPA, which may yield faster results (minutes vs. 24-72h) than the immunodiagnosis assays frequently used in bovine brucellosis. In addition, NH tracer produces similar or better performance results than the conventional OPS tracer, using the FPA in goat sera samples.     

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.     

Enzyme-linked immunosorbent assay for Brucella ovis specific antibody in ram sera

An enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella ovis specific antibody in ram serum was compared with the currently employed complement fixation test (CFT). Rabbit anti-sheep IgG coupled to horseradish peroxidase was used as the antibody-enzyme conjugate and 2,2'-azino-di[3-ethylbenzthiazolin sulphonate (6)] as the substrate. The ratio of the optical density at 414 nm for positive and negative control sera (P/N ratio) was used to optimise the parameters of the test. Ram serum samples (16,527) were tested using ELISA and CFT (warm and cold) over a one year period. The ELISA was more sensitive and provided a more reliable measure of B ovis specific antibody than did the CFT. Implications of employing ELISA as the sole test in an eradication scheme are discussed.     

Validation of the Fluorescence Polarisation Assay (FPA) for the serological diagnosis of brucellosis

In order to evaluate suitability of Fluorescence Polarisation Assay (FPA) for serological Brucella diagnostic, 1739 samples of sera from cattle, pigs, sheep and goats (65 Brucella-positive, 960-negative and 714 false-positive sera) were investigated at a dilution of 1:10. The cut-off was adjusted by means of ROC analysis. Furthermore, the serum samples were examined for Brucella antibodies using SAT, CFT and ELISA and the results were evaluated regarding sensitivity and specificity. FPA, SAT, CFT and ELISA attained a sensitivity of 92.3, 98.5, 84.6 and 86.2%. In comparison, specificity varied with 87.8, 72.6, 92.5 and 85.8%, respectively. Accordingly, FPA is a suitable test for serodiagnosis of brucellosis.