Evaluation of the enzyme-linked immunosorbent assay for detecting Brucella abortus antibodies.

The specificity of enzyme-linked immunosorbent assays (ELISA) corresponds to conventional methods for detecting brucella antibodies in bovine serum. The ELISA test detected brucella antibodies early in only 12.5% of the cattle sera tested. Also, the sensitivity of ELISA was comparable to complement-fixation and Rivanol methods, but less sensitive than the standard tube agglutination method.     

Evaluation of serologic and cellular immune responses of cattle to a nonlipopolysaccharide antigen from Brucella abortus

Cows naturally infected with Brucella abortus developed antibody (Ab) responses to a nonlipopolysaccharide antigen (NLA) purified from B abortus strain 1119-3. Sera from strain 19-vaccinated cows did not have detectable amounts of Ab. Weak lymphoproliferative responses to NLA were observed in blood mononuclear cell suspensions obtained from infected cows. There was no evidence of NLA-specific lymphoproliferation in cell suspensions from healthy cows. Nonlipopolysaccharide antigen binding to bovine blood mononuclear cells was observed by antigen-consumption assays and direct binding of radiolabeled antigen. Cells from infected cows bound less NLA than did cells from healthy cows when assays were conducted with intact blood mononuclear cell preparations (monocytes plus lymphocytes). Monocytes obtained from any group did not bind NLA. Purified B lymphocytes from infected and healthy vaccinated cows bound about 3 times more NLA than did T lymphocytes, but there were no apparent differences between the 2 groups in extent of binding. Results of the study indicate that bovine lymphocytes have binding sites for a NLA purified from B abortus strain 1119-3.     

Enzyme-linked immunosorbent assay for detecting antibodies to Brucella abortus in bovine milk and serum

An enzyme-linked immunosorbent assay (ELISA) method was evaluated for the detection of antibodies to Brucella abortus in cows milk. Milk samples from seropositive or -negative cows were sed to determine the distribution of absorbance values to classify milk as ELISA positive or ELISA negative. Brucella abortus was isolated from milk samples from 10 (45%) of the 22 cows whose milk and serum were ELISA positive. The ELISA was evaluated and determined to be an appropriate method for detecting antibodies to B abortus in bovine milk.     

Experimental infection of dogs with Brucella abortus: effect of exposure dose on serologic responses and comparison of culture methods

Two studies of experimentally induced Brucella abortus infections in dogs are reported. Twenty dogs in experiment 1 were fed approximately 4.8 X 10(10) colony forming units of B. abortus strain 2308 (BA 2308), and 11 dogs in experiment 2 were fed approximately 3.4 X 10(14) BA 2308. Serum samples from each infected dog and noninfected control were tested on the day of infection and at weekly and biweekly intervals post-infection (PI) for antibodies to B. abortus. The standard tube agglutination (STA) and rivanol (RIV) mean log-titers of the infected dogs in the 2 experiments were compared statistically on the day of infection and on PI days 7, 14, 21, 35, 42 and 49. The STA and RIV titers of the infected dogs in experiment 2 were significantly higher than were those of the dogs in experiment 1. Brucella abortus was isolated from 29 infected dogs in experiments 1 and 2 including 10 dogs, which were seronegative when cultured.     

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 review of enzyme immunoassay for detection of antibody to Brucella abortus in cattle

Enzyme immunoassay has gained wide acceptance for serological diagnosis of bovine brucellosis because of its ability to detect antibody of all isotypes unlike the conventional tests. The indirect enzyme immunoassay, however, presents several parameters that require careful analysis. These parameters include the choice of antigen and antiglobulin-enzyme conjugate reagents for use in the assay, dealing with the large amount of data the semi-automatic or automatic assay can generate and the inter- and intralaboratory standardization and quality control. This review considers the various methods described in the literature and, briefly, how some of the problems have been overcome or how they might be dealt with.     

Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed. This assay was different from previously developed CELISAs in that the detection reagent used was a recombinant combination of the receptor portions of protein A and protein G, labelled with horseradish peroxidase. This eliminates the need for polyclonal anti-mouse-enzyme conjugate reagents for detection thus allowing for true standardization. The assay utilized a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3 but which did not react with protein A/G. This monoclonal antibody was used to compete with antibody in the bovine test serum. Binding of bovine antibody to the smooth lipopolysaccharide antigen was then measured directly with the protein A/G enzyme conjugate. In this case, development of colour in the reaction was indicative of the presence of bovine antibody. The performance characteristics, sensitivity, specificity and exclusion of B. abortus S19 vaccinated animals, of the assay were very similar to those of the classical CELISA.     

Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves.

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0-3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.     

Comparison of three culture techniques for the isolation of Brucella abortus from bovine supramammary lymph nodes

Three different culturing techniques were compared and evaluated to determine the most effective method for isolating Brucella abortus from bovine supramammary lymph nodes (SM's). In method I, the SM was sliced in half, and the inner surface was minced finely with a sterile scalpel. The minced surface was spread onto the agar surface of 4 selective media. In method II, the SM was cut into small pieces and placed in a bag with a volume of phosphate-buffered saline equal to the volume of the lymph node. The bag was placed in a laboratory blender and the SM was macerated for 5 min. The tissue suspension was spread with a sterile cotton swab onto the agar surface of 4 selective media. In method III, the SM was processed in the laboratory blender. One milliliter of the suspension was pipetted into a flask of biphasic medium, and 2 ml of the suspension was pipetted into another flask of biphasic medium. A total of 626 SM's from 285 cows were cultured. Brucella abortus was isolated from 149 (52.3%) cows by 1 or more methods. Brucella abortus was isolated from 136 cows by method I. 137 cows by method II, and 86 cows by method III. Nine (3.2%) cows were positive by method I only, 11 (3.9%) cows by method II only, and 2 (0.7%) cows by method III only. The isolation rate for method III was significantly lower than for method I or II. There was no significant difference between methods I and II.     

Bovine brucellosis: evaluation of field sera by a competitive and superimposable ELISA utilising a monoclonal antibody against Brucella abortus lipopolysaccharide

With the aid of a horseradish peroxidase (HRP) tagged monoclonal antibody against smooth lipopolysaccharide from Brucella abortus (Bruce 1), a competitive and superimposable ELISA test procedure for bovine brucellosis has been evaluated for its ability to discriminate between Strain 19-vaccinated (S19-Vacc) and Biotype 1-infected (B1-Inf) cattle. In the competitive assay, all sera from S19-Vacc animals competed effectively against HRP-Bruce 1 (low HRP activity), while 10 out of 40 B1-Inf animals competed less effectively with Bruce 1 (high HRP activity). Successful competition by cattle antibodies would result in an increased proportion of cattle Igs binding to the assay antigen. This was confirmed by superimposing an alkaline phosphatase conjugated rabbit anti-cattle Ig after the competitive ELISA had been completed. With the superimposable assay, alkaline phosphatase activity was correspondingly high for S19-Vacc animals, and low for 36 out of 40 B1-Inf animals. The superimposable ELISA had therefore improved the discriminatory capabilities of the assay procedure from 75% to 90%.     

Development of an enzyme-linked immunosorbent assay for detecting antibodies in sera of Brucella suis-infected swine.

An enzyme-linked immunosorbent assay was developed using a heat-killed Brucella suis antigen for detecting antibodies in the sera of swine from which B. suis was isolated. Optimal enzyme-linked immunosorbent assay reactions were obtained using heat-killed B. suis antigen at a concentration comparable to McFarland Standard No. 1. Statistically significant differences were observed in the enzyme-linked immunosorbent assay results of 40 animals from which B. suis was isolated and the results for 48 noninfected swine at serum dilutions of 1:25 and 1:50 (P < 0.0001). The enzyme-linked immunosorbent assay is a rapid reproducible test which can be readily automated that appears to have practical value for screening large numbers of breeding and slaughter swine for brucellosis.     

Serological survey of Brucella abortus antibody prevalence in the one-humped camel (Camelus dromedarius) from eastern Sudan

A five year investigation of Brucella antibody prevalence in camel sera was conducted in 1502 one-humped camels of both sexes and different ages. The average (mean +/- SD) incidence rate of positive results was 6.95 +/- 1.55%. Among adult one-humped camels, the rate was 4.94 +/- 2.51% in males and 13.76 +/- 4.41% in females. Juvenile one-humped camel calves showed a 0% incidence rate in males and a 1.82 +/- 3.64% in females. Antibodies against Brucella abortus were prevalent in one-humped camel sera throughout the five years of the survey with incidence rates of 6.54, 5.79, 9.32, 5.03 and 8.06%, respectively from 1985 to 1989.     

Duration of strain 2308 infection and immunogenicity of Brucella abortus lipopolysaccharide in five strains of mice

A study was conducted to compare immunogenicity of a Brucella abortus lipopolysaccharide (LPS) and the duration of infection in 5 strains of mice. Mice of strains CBA/NJ, BALB/c, CD-1, C3H/HeN, and C3H/HeJ were allotted into 2 large groups (vaccinated with proteinase K-treated LPS or nonvaccinated) and 6 subgroups based on the intervals between challenge exposure to B abortus strain 2308 and the week the response data were obtained. Criteria used in comparing responses between the various strains of mice as well as between vaccinated and nonvaccinated mice were splenomegaly, colony-forming units (CFU) from spleens, and antibody titers. Responses were evaluated at 1, 2, 3, 5, 8, and 12 weeks after challenge exposure. Results indicated that all strains of mice became infected and maintained infection throughout the 12-week period, the percentages of mice infected were significantly (P less than 0.05) less in vaccinated mice for the first 5 weeks after challenge exposure, and there were no direct correlations between increased immunoglobulins (IgM and IgG titers) and reduction in CFU. Vaccinated mice of strains BALB/c, CD-1, C3H/HeN, and C3H/HeJ had increased titers when challenge exposed and also had significantly (P less than 0.05) smaller spleens and lower CFU. Vaccinated CBA/NJ mice did not have marked antibody titers. The overall results indicated that vaccination with LPS offers some initial protection against B abortus strain 2308 infection, but this protection disappears gradually and in various degrees in the 5 strains of mice studied.     

A comparison of six methods used for detecting pregnancy in sheep

The speed and accuracy of 4 ultrasonic devices (Scanopreg, Pregmatic 3, Medata external probe and Medata rectal probe) were compared with udder scoring and harnessed vasectomised rams as methods of pregnancy diagnosis in 177 ewes, 59 of which were non-pregnant. When using the Scanopreg in ewes of greater than 50 days gestation, accuracy in pregnant ewes averaged 98% and in non-pregnant ewes 87%. The Pregmatic 3 was 97% accurate in pregnant ewes after 50 days gestation and was 96% accurate in non-pregnant ewes. Ewes were examined at between 85 and 185 per hour using these devices. The Medata external probe was 99% accurate in pregnant ewes of greater than 110 days gestation but less accurate in early gestation. This instrument averaged 89% accuracy in non-pregnant ewes and an examination rate of 29 to 35 ewes per hour was achieved. The Medata rectal probe averaged 85% accuracy in pregnant ewes after 70 days gestation. Accuracy averaged 94% in non-pregnant ewes. Ewes were examined at between 35 and 59 per hour using this device. After 130 days gestation 84% of ewes had firm, obviously enlarge udders and another 14% had slight to moderate udder development. After 130 days gestation thick, clear udder secretions were expressed from 18% of ewes and thick, milky udder secretions were expressed from 8%. Harnessed vasectomised rams raddled one of 118 pregnant ewes (an accuracy in pregnant sheep of 99%) and raddled 53% of non pregnant ewes. It was concluded that the ultrasonic pregnancy testing devices have a commercial role, especially as aids to decision making.(ABSTRACT TRUNCATED AT 250 WORDS)     

两种检测免疫猪伪狂犬病血清抗体的方法比较

用2批伪狂犬病活疫苗分别免疫40～50 kg的健康敏感猪,每批疫苗免疫4头,每头肌注1头份.分别于免疫10 d、20 d后采血,分离血清.采用中和试验和乳胶凝集试验(LAT)两种方法检测血清抗体.结果显示:免疫10 d和20 d后,伪狂犬病中和抗体效价和LAT效价的几何平均值(GMT)分别为9.8、2.0,11.7、2.0;43.5、4.8,61.8、5.3.免疫20 d后,血清中和抗体效价为免疫10 d后的4倍以上,而血清LAT效价为免疫10 d后的2.5倍左右.     

Comparison of an indirect ELISA with the Brucella milk ring test for detection of antibodies to Brucella abortus in bulk milk samples.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella abortus antibodies in bovine bulk milk samples was evaluated. About 31 individual milk samples from B. abortus infected cows were diluted into bulk milk from a brucellosis free herd. Individual milk samples obtained from 96 negative or positive herds to ELISA or Brucella ring test (BRT), were tested by ELISA. All positive cows were bled and serum samples were tested by the complement-fixation test (CFT) which was considered the definitive test. A herd was considered infected if at least, one cow was positive in the CFT. Four samples were negative in the BRT at the dilution 1:10 but positive in the ELISA. For samples positive in both tests, BRT titers ranged from 1:10 to 1:480 while ELISA titers ranged from 1:10 to 1:3200.Using bulk milk samples, the sensitivity of the ELISA (98.1%) was higher than the BRT (72.2%) but the specificity of BRT (90.5%) was not statistically different (P=1.0) from the ELISA (88.1%). The implications of the results for brucellosis control are discussed.     

Enzyme-linked immunosorbent assays for detecting antibody to Rickettsia australis in sera of various animal species

New endemic areas of spotted fever-like rickettsial disease have been found in south-eastern Australia (Gippsland, Victoria and Flinders Island, Tasmania). The rickettsia responsible is currently unknown although it may be Rickettsia australis. To investigate serological evidence of rickettsial exposure in various wild animal species, a competitive ELISA was developed which detected antibodies to R. australis. It was based on inhibition of an indirect ELISA detecting antibody to R. australis in guinea pig sera. Pre- and post-infection sera from 2 dogs, 2 rabbits, 5 mice and 6 rats, experimentally infected with R. australis, were tested by competitive ELISA. The results showed that all pre-infection sera were negative and all post-infection sera positive for antibody to R. australis. To test the utility of the competitive ELISA for detecting natural rickettsial infection in non-laboratory animals, 51 dog sera, negative for rickettsial antibody by immunofluorescence (IF) and 20 IF positive dog sera (collected from various locations on the east coast of Australia) were tested. Compared to the IF test the competitive ELISA was 90% sensitive and 96% specific. This new test has potential for detecting antibody to R. australis in the sera of different wild animal species.     

Comparison of serologic tests for detection of Brucella infections in cattle and water buffalo (Bubalus bubalis)

OBJECTIVE: To estimate sensitivity and specificity of 4 commonly used brucellosis screening tests in cattle and domestic water buffalo of Trinidad, and to compare test parameter estimates between cattle and water buffalo. ANIMALS: 391 cattle and 381 water buffalo. PROCEDURE: 4 Brucella-infected herds (2 cattle and 2 water buffalo) and 4 herds (2 of each species) considered to be brucellosis-free were selected. A minimum of 100 animals, or all animals > 1 year of age, were tested from each herd. Serum samples were evaluated for Brucella-specific antibodies by use of standard plate agglutination test (SPAT), card test (CT), buffered plate agglutination test (BPAT), and standard tube agglutination test (STAT). A Bayesian approach was used to estimate sensitivity and specificity of diagnostic tests without the use of a gold standard, assuming conditional independence of tests. RESULTS: Sensitivity and specificity estimates in cattle, respectively, were SPAT, 66.7 and 98.9; CT, 72.7 and 99.6; BPAT, 88.1 and 98.1; and STAT, 80.2 and 99.3. Corresponding test estimates in water buffalo, respectively, were SPAT, 51.4 and 99.3; CT, 90.4 and 99.4; BPAT, 96.3 and 90.7; and STAT, 75.0 and 98.8. Sensitivity of the CT and specificity of the BPAT were different between cattle and water buffalo with at least 95% probability. CONCLUSIONS AND CLINICAL RELEVANCE: Brucellosis serologic test performance varied by species tested, but BPAT had the highest sensitivity for screening cattle and water buffalo. Sensitivity and specificity of more than 2 screening tests can be estimated simultaneously without a gold standard by use of Bayesian techniques.