A comparison of five serological tests for bovine brucellosis.

Five serological assays: the buffered plate antigen test, the standard tube agglutination test, the complement fixation test, the hemolysis-in-gel test and the indirect enzyme immunoassay were diagnostically evaluated. Test data consisted of results from 1208 cattle in brucellosis-free herds, 1578 cattle in reactor herds of unknown infection status and 174 cattle from which Brucella abortus had been cultured. The complement fixation test had the highest specificity in both nonvaccinated and vaccinated cattle. The indirect enzyme immunoassay, if interpreted at a high threshold, also exhibited a high specificity in both groups of cattle. The hemolysis-in-gel test had a very high specificity when used in nonvaccinated cattle but quite a low specificity among vaccinates. With the exception of the complement fixation test, all tests had high sensitivities if interpreted at the minimum threshold. However, the sensitivities of the standard tube agglutination test and indirect enzyme immunoassay, when interpreted at high thresholds were comparable to that of the complement fixation test. A kappa statistic was used to measure the agreement between the various tests. In general the kappa statistics were quite low, suggesting that the various tests may detect different antibody isotypes. There was however, good agreement between the buffered plate antigen test and standard tube agglutination test (the two agglutination tests evaluated) and between the complement fixation test and the indirect enzyme immunoassay when interpreted at a high threshold. With the exception of the buffered plate antigen test, all tests were evaluated as confirmatory tests by estimating their specificity and sensitivity on screening-test positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validation of enzyme-linked immunosorbent assays for the diagnosis of bovine brucellosis

The purpose of this study was to evaluate the performance of the indirect enzyme immunoassay (IELISA) and the competitive enzyme immunoassay (CELISA) for the diagnosis of bovine brucellosis in comparison to conventional serological tests routinely used in Argentina. Serum samples (n = 3500), from Brucella-free herds, from vaccinated cattle and from naturally infected cattle, were tested by the following tests: buffered antigen agglutination test (BPAT), rose bengal test (RBT), 2-mercaptoethanol test (2-ME), complement fixation test (CFT), IELISA and CELISA. Sensitivity and specificity of the BPAT, RBT, IELISA and CELISA were determined relative to the 2-ME and the CFT. The CELISA was considered suitable for eliminating most serological reactions of vaccinated animals and was more specific than the other tests. The results indicate the potential use of the CELISA as a complementary assay in the brucellosis control and eradication program in Argentina and other countries, where Brucella abortusstrain 19 vaccination is mandatory.     

Use of the brucellosis card test for screening cattle in Saskatchewan.

One group of 28,714 bovine sera were tested by both the brucellosis tube serum agglutination test and the brucellosis card test. The tube serum agglutination test confirmed 99.8% of the negative brucellosis card test results. The brucellosis card test identified 63% of the tube serum agglutination test reactors. In a second group of 496 sera reacting to either the tube serum agglutination test, complement fixation test, plate serum agglutination test or acid antigen serum agglutination test the brucellosis card test identified 99.1% of the complement fixation test positive sera and 91.3% of the sera reacting to any of the other serological tests. The brucellosis card test showed satisfactory agreement with both the complement fixation test and tube serum agglutination test. It appears to be a useful screening test in operations involving large numbers of animals since under these conditions the reactors can be quickly identified and isolated.     

A comparison of standard serological tests for the diagnosis of bovine brucellosis in Canada.

Six agglutination and two complement fixation tests were compared with respect to specificity, sensitivity and relative sensitivity for the serodiagnosis of bovine brucellosis. Based on 1051 sera from brucellosis free herds, the specificity of the tests was 98.9% for the buffered plate antigen test (BPAT), 99.2% and 99.3% for the standard tube and plate agglutination tests (STAT and SPAT), respectively, and 99.8% for the 2-mercaptoethanol test (2MET). On this small sample, the rose bengal plate test (RBPT), card test (CARD) and the complement fixation test (CFT) correctly classed all sera as negative. On a sample of 167 culture positive cattle, the sensitivities of the tests were CFT: 79.0%, BPAT: 75.4, RBPT: 74.9%, CARD: 74.3%, SPAT: 73.1%, STAT: 68.9%, and 2MET: 59.9%. All tests combined detected only 82% of these infected cattle. Analysis of the relative sensitivity of the six agglutination tests gave the following ranking: BPAT greater than RBPT greater than CARD greater than SPAT greater than STAT. The 2MET ranked between the BPAT and RBPT or between the RBPT and CARD depending on the analysis used. The use of the BPAT as a screening test is recommended provided that a test of high specificity and sensitivity such as the CFT is used to confirm screening test reactions.     

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.     

Fluorescence polarization assay for the diagnosis of bovine brucellosis: adaptation to field use

A fluorescence polarization assay (FPA) was used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. abortus) with sera from animals with confirmed (bacteriologically) infection. A cut-off value between negative and positive values was initially established to be 87.2mP. This value was changed to 95mP to increase assay specificity without loss of sensitivity when testing blood samples from negative animals. The FPA technology was applied to whole blood samples in the field and to stored whole blood samples using two diluent buffers. Relative sensitivity and specificity values for the FPA performed in the field, based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95.3 and 97.3%, respectively. However, to obtain maximum sensitivity and specificity, a cut-off value of 105mP was determined for fresh whole blood samples. The relative sensitivity and specificity values of the FPA when testing stored whole blood samples were 100% each using a 95mP cut-off.The usefulness of the FPA for testing whole blood samples in the field was demonstrated.     

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.     

Diagnosis of brucellosis by serology

Serological diagnosis of brucellosis began more than 100 years ago with a simple agglutination test. It was realized that this type of test was susceptible to false positive reactions resulting from, for instance, exposure to cross reacting microorganisms. It was also realized that this test format was inexpensive, simple and could be rapid, although results were subjectively scored. Therefore, a number of modifications were developed along with other types of tests. This served two purposes: one was to establish a rapid screening test with high sensitivity and perhaps less specificity and a confirmatory test, usually more complicated but also more specific, to be used on sera that reacted positively in screening tests. This led to another problem: if a panel of tests were performed and they did not all agree, which interpretation was correct? This problem was further compounded by the extensive use of a vaccine which gave rise to an antibody response similar to that resulting from field infection. This led to the development of an assay that could distinguish vaccinal antibody, starting with precipitin tests. These tests did not perform well, giving rise to the development of primary binding assays. These assays, including the competitive enzyme immunoassay and the fluorescence polarization assay are at the apex of current development, providing high sensitivity and specificity as well as speed and mobility in the case of the fluorescence polarization assay.     

National Swine brucellosis survey

A national survey was conducted in 1985 to investigate the brucellosis status of the Canadian swine herd. Serum samples were collected from cull sows slaughtered over a forty week period in 1985; 15,707 samples were suitable for brucellosis testing, and 48 (0.31%) gave some degree of reaction on the buffered plate agglutination screening test. All 48 samples were negative on the 2-mercaptoethanol and modified complement fixation test. We therefore conclude that the Canadian swine herd remains free of brucellosis.     

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.     

Experience with simple ELISA test systems for Brucella serology in cattle, sheep and goats

The objective of this work was to use the ELISA technique for the serological surveillance for freedom of brucellosis of cattle, sheep and goats. By comparing 28 cattle sera taken after a brucellosis outbreak, 15 bovine sera supplied by the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) and 497 serum slow agglutination test (SSAT) and complement fixation test (CFT) negative bovine sera from herds officially declared free of brucellosis, the ELISA technique not only shows higher sensitivity as compared to SSAT and CFT but also distinguishes clearly between positive and negative reactions. The serological comparison by SSAT, CFT and ELISA of 615 cattle, 624 sheep and 630 goat sera from herds acknowledged as brucellosis free showed equivalent specificities for both CFT and ELISA. The specificity of the SSAT was much lower, 81.1% in cattle and 96.2% in goat sera. The examination of 5796 cattle, 1337 calf, 5031 sheep and 1796 goat sera demonstrates the advantage of the ELISA technique as routine method. The possible application of the ELISA technique as a screening method for serological brucellosis tests in sheep, goats and possibly also in pigs is discussed.     

Diagnostic sensitivity and specificity of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams.

An enzyme-linked immunosorbent assay (ELISA) for antibody to Brucella ovis was compared with a standard complement fixation test. Sera of 176 rams from uninfected flocks gave 175 negative and one suspect ELISA reaction (diagnostic specificity 99.4%) whereas the complement fixation test yielded 167 negative, seven suspect and two anticomplementary reactions (diagnostic specificity of 96.0%). Diagnostic sensitivity was evaluated on sera of 79 rams from which B. ovis had been isolated. The ELISA showed 75 positive and four suspect reactions, while complement fixation test revealed 64 positive, 13 suspect and two negative results. Considering both positive and suspect reactions, the diagnostic sensitivity was 100% for ELISA and 97.5% for complement fixation test. The ELISA method was considered more specific, more sensitive and technically more advantageous than complement fixation test as a serodiagnostic test for B. ovis infection in rams.     

Development and validation of a competitive ELISA kit for the serological diagnosis of ovine, caprine and bovine brucellosis

A competitive ELISA (Brucella-Ab c-ELISA) was standardized and validated for the detection of Brucella antibodies in cattle, sheep and goat sera using a monoclonal antibody (MAb 4B5A) produced against Brucella melitensis biotype 2. The specificity and sensitivity of the assay were 100% to a 67.5% cut-off point (B/Bo%). When compared with an indirect ELISA, the Brucella-Ab c-ELISA did not demonstrate cross-reactions when testing positive sera for antibodies to some Enterobacteriaceae. A comparison was made between the Brucella-Ab c-ELISA and the complement fixation and Rose Bengal tests. Results demonstrated that the Brucella-Ab c-ELISA is a valuable tool for the serological diagnosis of bovine and ovine/caprine brucellosis.     

Estimation of sensitivity and specificity of five serological tests for the diagnosis of porcine brucellosis

While serological tests are essential in surveillance and control programs of animal diseases, to date none of the common serological tests approved in the EU (complement fixation test or Rose-Bengal test) has been shown to be reliable in routine individual diagnosis of porcine brucellosis, and some more recent tests like ELISA have not been fully evaluated yet. In the absence of a gold standard, this study allowed the estimation of sensitivities and specificities of these tests with a Bayesian approach using Markov Chain Monte Carlo algorithms. The pig population that was tested included 6422 animals from Metropolitan France. Serum samples were collected from a large population of pigs, representative of European swine population and tested with five brucellosis serological tests: Rose-Bengal test (RBT), fluorescence polarization assay (FPA), indirect ELISA (I-ELISA) and two competitive ELISAs (C-ELISA). The sensitivity and the specificity of each test were estimated. When doubtful results were excluded, the most sensitive and specific test was C-ELISA(2) (Se C-ELISA(2)=0.964, [0.907; 0.994], 95% credibility interval (CrI); Sp C-ELISA(2)=0.996, [0.982; 1.0], 95% CrI). When doubtful results were considered as negative, C-ELISA(2) was still the most sensitive and specific test (Se C-ELISA(2)=0.960, [0.896; 0.994], 95% CrI and Sp C-ELISA(2)=0.994, [0.977; 0.999], 95% CrI). The same conclusions were reached when doubtful results were considered as positive (Se C-ELISA(2)=0.963, [0.904, 0.994], 95% CrI and Sp C-ELISA(2)=0.996, [0.986; 1.0], 95% CrI).     

The use of divalent cation chelating agents (EDTA/EGTA) to reduce non-specific serum protein interaction in enzyme immunoassay

An indirect enzyme immunoassay (ELISA) for detection of bovine antibody toBrucella abortus was modified by the addition of divalent chelating agents to the serum diluent. This addition resulted in an increase in specificity from 96.0% in the regular assay to 99.4% in the modified procedure. Of the 15 715 sera initially tested by the indirect ELISA, 691 that had given positive reactions were selected for retesting in the indirect ELISA with EDTA/EGTA added. The buffered plate antigen test (BPAT) correctly identified 98.6% of the samples as negative. The addition of chelating agents did not alter the sensitivity of the indirect ELISA, which correctly classified 609 sera from animals from whichB. abortus had been isolated as positive. The sensitivity of the BPAT was 97.8%.Abbreviations ABTS 2,2-azinobis(3-ethylbenzthiazoline sulphonic acid) - BPAT buffered plate antigen test - EDTA ethylenediaminetetraacetic acid disodium salt - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ELISA enzyme-linked immunosorbent assay - IgG₁ immuno- globulin G₁ - IgM immunoglobulin M - Tris tris(hydroxymethyl)aminomethane     

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle–protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar’s analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.     

Detection of antibody to Bordetella avium using a particle concentration fluorescence immunoassay (PCFIA).

A fluorescent assay for detection of antibody to Bordetella avium was developed and evaluated against current serological techniques. Bordetella antigen was coated on submicron polystyrene beads and used in an antibody sandwich technique with commercially available goat anti-turkey fluorescein-labeled IgG. Positive control sera were taken from turkeys from which B. avium was isolated. Negative control sera were from turkeys from which no B. avium was isolated and which tested negative by agglutination and enzyme immunoassay. A commercial fluorescence concentration analyzer was used to quantitate the epifluorescence in a special 96-well plate designed to minimize background fluorescence. The particle concentration fluorescence immunoassay was found to be as sensitive as the enzyme immunoassay, with good reproducibility. It required less time and smaller quantities of sample and reagents.     

A comparative assessment of culture and serology in the diagnosis of brucellosis in dairy cattle

To investigate the usefulness of culture for the confirmation of brucellosis in cattle, a comparison of culture and serology was undertaken on 248 animals in four dairy herds where the disease was active. Paired supramammary (SM), retropharyngeal (RP), and internal iliac (IL) lymph nodes were cultured, and five serological tests were deployed: the microserum agglutination test (MSAT), complement fixation test (CFT), the indirect (iELISA) and competitive ELISA, and the fluorescence polarisation assay (FPA). Brucella abortus was isolated from 86.8% of animals on combined culture of all three lymph nodes. Individually, the highest isolation rate was from the RP (90.5% of culture positives). Of culture positive animals, 13.7% and 6.2% were positive from the RP and SM alone, respectively.Approximately half of the positive cultures yielded <10 colonies/culture plate. Although 80.9% of animals were positive in at least one serological test, only 45.2% were positive in all five. For culture-positive animals, the MSAT was the most sensitive test (71.8%). Of the culture-negative animals 67.7% were positive in at least one test, while 12.9% were positive in all five. Titres were higher in animals culture-positive from the SM, and there was a direct correlation between higher titres and higher colony counts in SM cultures. Only 8.9% of animals were both culture-negative and seropositive (in at least one test), while 16.5% were culture-positive and seronegative in all five tests. The results highlight and validate the sensitivity of bacteriological culture in confirming a diagnosis of bovine brucellosis. While the MSAT and FPA were the most sensitive serological tests, a significant percentage of infected animals were undetectable using these standard serological assays.     

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.     

Serological cross-reactivity of porcine reference antisera to Mycoplasma hyopneumoniae, M. flocculare, M. hyorhinis and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirect hemagglutination tests.

The enzyme-linked immunosorbent assay (ELISA) indicated significant cross-reactivity between the antigens of Mycoplasma hyopneumoniae ( HyoP ) and M. flocculare (Floc), another porcine mycoplasma of wide distribution but uncertain pathogenic significance, when porcine antisera of each specificity were tested against HyoP antigen. The titers of the anti-Floc sera ranged from threefold to 13-fold less than the titer of the anti- HyoP reference serum at different times after immunization. These values ranged from onefold less than to fourfold greater than the minimal positive titer of 80. The antisera to the other porcine mycoplasmal antigens [i.e. M. hyorhinis ( HyoR ) and M. hyosynoviae ( HyoS )] reacted less strongly to HyoP antigen but titers only slightly less than to slightly greater than the minimal positive titer were noted for some sera. Cross-reactivity was also detected by the complement fixation test, although the titers for this test were generally lower than for the ELISA, presumably reflecting lower sensitivity of the complement fixation test. Positive indirect hemagglutination titers to HyoP antigen were also observed for both anti-Floc sera obtained at one or more times during the immune response. With two exceptions (one anti- HyoR serum with a complement fixation titer of 16 and one anti- HyoR serum with an indirect hemagglutination titer of 10), none of the anti- HyoR or anti- HyoS sera had detectable indirect hemagglutination or complement fixation titers to HyoP antigen at any time after immunization. The levels of cross-reactivity detected by the complement fixation test and indirect hemagglutination and, especially, the ELISA would be of significance for the development of any practical sero-diagnostic test for mycoplasmal pneumonia of swine.