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.

A serological comparison of complement fixation reactions using Brucella abortus and B. melitensis antigens in B. abortus infected cattle.

Brucella abortus and B. melitensis antigens were used in parallel on the National Standard Brucella abortus antiserum and on field sera coming from cattle where practically exclusively B. abortus biotypes 1 and 2 have been isolated over the last 11 years. With the National Standard serum the titres to B. melitensis were consistently lower than those to B. abortus antigen. Most were 1 dilution (twofold) lower. Although a similar trend was seen with the field sera, there were 7/346 sera which had twofold or higher titres to B. melitensis antigen. Although this may be due to the vagaries of the test it also warrants closer investigation of the animals concerned to see whether M-antigen predominant Brucella biotypes are possibly present. The use of the dual antigens could identify herds which are infected only with A-antigen predominant brucellae but would not be reliable for classifying individual animals.

An ELISA using an SDS extract of Brucella abortus strain 99 as antigen to detect B. abortus antibodies in cattle sera.

An ELISA was developed using an SDS extract of Brucella abortus as antigen to detect antibodies in cattle sera. The antigen was stable at 4 degrees C for at least 4 years and although it gave optimal results at a 1/4,000 dilution it could detect reactors at a 1/32,000 dilution. Based on comparative CF tests on 430 sera from negative herds and 187 sera from positive herds a reading of 0,07 or less using a 492 nm filter could be considered a negative reaction. The ELISA could be a useful test to supplement the CF test but further evaluation is still required.

A rapid microtitration serum agglutination test for the detection of contagious equine metritis antibodies.

A microtitration serum agglutination test, based on that used for brucellosis, has been developed to detect antibodies in the sera of horses exposed to the contagious equine metritis (CEM) organism. Two known positive sera were tested 100 times in 15 separate tests. The results were reproducible to within a twofold range. The test is capable of being carried out within 100 min.

A short, reliable, highly reproducible complement fixation test for the serological diagnosis of contagious equine metritis.

A complement fixation test, using round-bottomed microtitration plates and an 8 channel microdiluter, based on that used for brucellosis by Herr, Huchzermeyer, Te Brugge, Williamson, Roos & Schiele, 1985, has been developed for use on the sera of horses to detect antibodies to the contagious equine metritis organism. The results with 2 known positive sera tested 116 times in 27 separate tests were reproducible for the most part within a twofold range. They seldom exceeded these limits and never exceeded a fourfold range. The test itself is capable of being carried out within 90 min. The test was slightly more sensitive when sera were inactivated in a hot air oven for 50 min at 58 degrees C, as compared to inactivation at 62 degrees C in a water-bath for 50 min. There were no false negative or false positive reactions and no anticomplementary activity in the sera tested.