Serological differentiation of Brucella-vaccinated and -infected domesticated animals by the agar gel immunodiffusion test using Brucella polysaccharide in mongolia

To investigate Brucella infection in cattle, sheep, goat, reindeer and yak in Mongolia, serological reactions of Brucella-infected and -vaccinated domestic animals were compared by the agar gel immunodiffusion (AGID) test with a polysaccharide (poly-B) of the B. Abortus strain S-19. The sensitivity and specificity were compared with conventional serological tests that are commonly used in Mongolia, such as the rose Bengal test, the tube agglutination test and the compliment fixation test. A total of 73.3, 100, 100, 95.8 and 61.9% of the sera from suspected cattle, yak, goat, sheep and reindeer, respectively, that were positive in the compliment fixation test, were also positive in the AGID test. Sera from vaccinated cattle, sheep and goat were positive over 90% by conventional tests 3 months after vaccination, but were negative by the AGID. These results suggest that the AGID test may be useful to differentiate infected and vaccinated animals in the field.     

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19 and in cattle infected with Brucella abortus field strain.

Cell-mediated immune responses in cattle adult-vaccinated with Brucella abortus strain 19, cattle infected with B abortus field strain, and nonexposed cattle were studied by an in vitro lumphocyte-stimulation test (LST). Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, and results were assayed for [3H]thymidine incorporation into DNA by liquid scintillation spectrometry. Serotests and bacteriologic isolation attempts were conducted simultaneously with LST. Lymphocytes from cattle infected with field strains had significantly (P = 0.01) higher specific lymphocyte-stimulation inexposed controls. The LST, the serum standard-tube agglutination test (STT), the Rivanol (RIV) test, and the complement-fixation (CF) test correctly classified cattle from which field strains and strain 19 of B abortus were isolated. The LST was negative in cattle vaccinated with B abortus strain 19 (nonshedding), but the three serotests had many false-positive reactions. The CF test had the least false-positive reaction, followed by the RIV test, and the STT was the least specific. Well before the three serotests became positive, the LST was positive in samples from some cattle during the incubation period of the infection. There was little or no correlation between cell-mediated immune responses (as measured by LST) and serum antibody responses (as measured by STT, RIV test, and CF test) in vaccinated but culture-negative cattle and in some nonvaccinated cattle during the incubation period.     

The bovine immune response to Brucella abortus IV. Studies with a double immunodiffusion test for antibody against A2.

A double immunodiffusion test for precipitins against Brucella antigen A2 was developed and applied to a variety of samples. The A2 precipitins were produced by a heifer infected with B. abortus strain 2308, cattle vaccinated with killed B. melitensis strain H38 or live B. abortus strain 19 and by a dog infected with B. canis. Precipitins were also detected in the second International Standard for anti-Brucella abortus serum, in several anti-B. canis sera and at low levels in one anti-B. ovis serum tested. Antisera produced in calves against Yersinia enterocolitica serotype 0:9 had no anti-A2 activity despite titers greater than or equal to 1/1024 and greater than or equal to 1/80 in standard Brucella agglutination and CF tests, respectively. The test for A2 precipitins lacked specificity as weak reactions were obtained with five of 295 sera from brucellosis-free herds. This test was relatively insensitive, detecting precipitins in only 16 of 24 sera from infected cattle and 27 of 54 sera positive by complement fixation and enzyme labelled antiglobulin tests performed with whole cell and smooth lipopolysaccharide antigens, respectively. The A2 precipitins were detected in nine sera from five cattle, in two infected herds, which were negative by agglutination and complement fixation tests.     

Comparison of an agar-gel immunodiffusion test with other serological methods for differentiating Brucella infected from vaccinated cattle.

An agar-gel immunodiffusion test was compared with other serological tests for detection of antibodies in the sera of 48 cattle vaccinated with Brucella abortus strain 19. Two hundred forty-two sera were tested over a 12 month period, and addition of positive reactions for each test resulted in totals of 170 for the card test, 74 for the standard tube-agglutination test, 71 for the 2-mercaptoethanol test, 64 for the dithiothreitol test and 22 for the agar-gel immunodiffusion test.     

Evaluation of polysaccharide, lipopolysaccharide, and beta-glucan antigens in gel immunodiffusion tests for brucellosis in cattle.

In Venezuela, 1,012 cattle sera were screened for their ability to precipitate Brucella melitensis 16M smooth-lipopolysaccharide (S-LPS), B melitensis B115 polysaccharide B (poly B), B abortus 1119-3 O-polysaccharide (PS), or B abortus 1119-3 cyclic 1,2 linked beta-D-glucan (beta-glucan) in an agar-gel immunodiffusion assay. These sera were previously classified as being Brucella abortus-infected, S-19-vaccinated, or negative after an assessment of historical records and results of 5 standard serologic tests. Most of the sera (85%) from infected cattle precipitated S-LPS, poly B, and PS. Serologic results for poly B and PS were identical. On the other hand, 13% of the sera from vaccinated cattle precipitated S-LPS, but none of these sera precipitated poly B or PS. It was concluded that purified PS can alternate with poly B as an antigen to differentiate sera of B abortus-infected from B abortus S-19-vaccinated cattle. None of these sera precipitated beta-glucan.     

Transient enhancement of the serum antibody response to Brucella abortus strain 19 in cattle treated with levamisole

Two experiments were done on 57 steers. These cattle were allotted to 8 groups (4 groups/experiment) and vaccinated with 1 to 3 X 10(9) colony-forming units of Brucella abortus strain 19. Cattle in 3 of the 4 groups/experiment were given 6 mg of levamisole/kg, subcutaneously, either at the time of vaccination (day 0), 7 days later, or at both times. Serum antibody titers to B abortus were measured sequentially for 28 days in experiment 1 and for 56 days in experiment 2, using the card test, Rivanol test, complement-fixation test, fluorometric immunoassay, and an enzyme-linked immunosorbent assay. In general, the highest mean antibody titers, as determined by all serologic tests, occurred in steers treated with levamisole at 7 days after vaccination or in those treated at the time of vaccination and 7 days later. By the card test on day 56, there was a significantly (P less than 0.05) greater number of seropositive cattle among those given levamisole 7 days after seropositive cattle among those given strain 19 alone. Simultaneous administration of strain 19 and levamisole did not alter antibody responses to B abortus.     

Differentiation by western blotting of immune responses of cattle vaccinated with Brucella abortus strain 19 or infected experimentally or naturally with virulent Brucella abortus

Brucella abortus strain 19 salt-extractable proteins fractionated by differential ammonium sulfate precipitation were used in a western blotting method to detect bovine immunoglobulin G antibodies to B. abortus. Sera from infected cattle and from cattle vaccinated with strain 19 and subsequently exposed to virulent B. abortus bound to a common group of antigens ranging in molecular weights from 31,000 to 45,000 daltons. Immunoglobulin G antibodies in sera from the latter group in addition also bound to antigens with molecular weights of 66,000 to 71,000 daltons. Some sera from cattle vaccinated when sexually mature reacted similar to those from infected cattle, while immunoglobulin G antibodies in sera from Brucella-free cattle and vaccinated calves did not bind to either group of antigens. In general, fractionation of the proteins by ammonium sulfate precipitation offered no advantage for detecting differences between groups of sera. Ammonium sulfate fraction 0 to 35% reacted with a larger number of sera from a naturally infected group than fraction 0 to 70%. Both fractions reacted equally well with sera from the other groups of cattle, while fractions 35 to 70% and 70 to 100% reacted poorly in this technique. The attractive feature of the blot is that sera from calfhood-vaccinated cattle did not react.     

The bovine immune response to Brucella abortus. II. Elimination of some sporadic serological reactions by chelation of divalent cations.

The standard agglutination tests for detecting antibody to Brucella abortus were modified by addition of chelating agents (EDTA and EGTA) to the antigens. Approximately 80% of "singleton" agglutination test reactions, negative on the diagnostic complement fixation test, obtained with cattle sera were eliminated while no decrease in titer was apparent when sera from B. abortus infected or vaccinated cattle were tested.     

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.     

A competition enzyme immunoassay for brucellosis diagnosis

Two monoclonal antibodies (MAbs) conjugated with horseradish peroxidase were used independently in a competitive enzyme immunoassay (cEIA) to detect Brucella specific antibodies in 1120 sera from Brucella-free cattle, 61 from cattle known to be infected with B. abortus, and 207 sera from vaccinated calves. The results were compared to those obtained in the complement fixation test (CFT). The cEIA with both MAbs proved to be more sensitive than the CFT because no false-negative results were obtained. In addition, discrimination between sera from infected and vaccinated animals was more evident.     

Brucella abortus-specific immunoglobulin isotypes in serum and vaginal mucus from heifers vaccinated with Brucella abortus salt-extractable proteins and challenge exposed with virulent Brucella organisms

Serum and vaginal Brucella-specific immunoglobulin isotypes (IgG1, IgG2, IgM, and IgA), obtained from 62 crossbred beef heifers vaccinated with Brucella abortus salt-extractable proteins and subsequently challenge exposed with B abortus S2308, were studied. Brucella-specific IgG antibodies and Brucella-specific immunoglobulin isotypes were quantitated by a fluorometric immunoassay. Serum and vaginal immunoglobulin responses were evaluated as a method of distinguishing infected from noninfected heifers. Rivanol precipitation, complement-fixation, buffered-antigen brucellosis tests and an ELISA were performed on sera. For immunoglobulin isotypes, vaccinated heifers had mean antibody responses higher than baseline mean antibody responses for at least 31 weeks after vaccination. After challenge exposure, significant differences (P greater than 0.05) were not detected between mean antibody responses of vaccinated and nonvaccinated heifers. Vaginal Brucella-specific antibody responses did not correlate with protection from disease. Vaginal Brucella-specific IgM was detected only at the time of abortion. Vaginal IgA appeared specific for identification of virulent B abortus infection. All serotests appeared adequate in distinguishing baseline titers from titers of heifers that had aborted and were considered bacteriologic culture-positive. Results of serotests neither consistently distinguished vaccinates from challenge-exposed cattle nor distinguished heifers that were challenge exposed, had aborted, and were considered bacteriologic culture-positive adequately from heifers that were challenge-exposed, had not aborted, and were considered bacteriologic culture-negative. Brucella-specific IgA appeared to be the most effective in distinguishing vaccinated heifers from challenge- exposed heifers and heifers that were challenge exposed and had aborted, from heifers that were challenge exposed and had not aborted. Brucella-specific serum IgA was detected up to 13 weeks after abortion.     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

Longitudinal studies of naturally acquired Brucella abortus infection in sheep.

Naturally acquired Brucella abortus infections were studied during consecutive pregnancies in eight sheep and in their lambs over a period of 40 months to evaluate epizootiologic aspects of natural infection in sheep. Brucella abortus was isolated from the ewes following 16 of 26 natural terminations of pregnancy: from 5 of 6 ewes in the first year, from six of eight ewes in the second year, from two of six ewes in the third year, and from three of six ewes in the fourth year. Vaginal swab samples and milk samples were the most consistent source of the brucella organisms. Brucella abortus was isolated from three ewes when standard tube test seroagglutination titers were less than 1:100. In contrast, results of supplemental tests (card, 2-mercaptoethanol, complement-fixation, and Rivanol) remained positive during the study. During the 40 months, B abortus was isolated from 4 of 4 aborted fetuses, 2 of 5 stillborn lambs, 10 of 37 living lambs, and as an indicator of continuing infection, from 6 of 12 lambs born during the fourth year. Although B abortus has a definite host preference for cattle, this study demonstrated that under appropriate management conditions, sheep may be naturally infected and may remain infected for more than 40 months. Epizootiologic evaluation of all factors, including husbandry practices and exposure potential, should be utilized in determining the need to test other species that may have been exposed to cattle infected with B abortus.     

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).     

Specific lymphocyte stimulation in cattle naturally infected with strains of Brucella abortus and cattle vaccinated with Brucella abortus strain 19.

Cell-mediated immune responses in cattle naturally infected with strains of Brucella abortus and in cattle vaccinated with B abortus strain 19 during calfhood were studied by an in vitro lymphocyte-stimulation procedure. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique, suspended in RPMI-1640 medium (1.5 X 10(6) lymphocytes/ml), cultured with B abortus-soluble antigen or phytohemagglutinin, and incubated for 6 days. Sixteen hours prior to termination of incubation, cultures were labeled with 1 muCi of [3H]thymidine (3HdT) and, after harvesting, assayed for 3HdT incorporation into DNA by liquid scintillation spectrometry. Lymphocytes from cattle with bacteriologically confirmed isolation of B abortus underwent a significantly higher lymphocyte stimulation with B abortus-soluble antigen than did cattle vaccinated with B abortus strain 19 during calfhood (P less than 0.005). Standard seroagglutination tests were conducted simultaneously with lymphocyte-stimulation tests, but there was no apparent correlation between levels of humoral antibodies and the cell-mediated immune responses as measured by in vitro specific lymphocyte stimulation.     

Comparison of an enzyme-linked immunosorbent assay using monoclonal antibodies and a complement fixation test for cattle vaccinated and infected with Brucella abortus

A competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody conjugated to horseradish peroxidase MA(A) and a complement fixation test (CFT) were applied to sera collected over a two-year period from 60 cattle challenged with Brucella abortus strain 544. Forty-eight of the cattle were previously vaccinated with B. abortus strain 19 (S19) or B. abortus strain 45/20 (45/20). After challenge 33 of the cattle remained uninfected and nine of the 27 infected cattle showed aberrant reactions by the CFT. The performance of the MA(A) ELISA was as follows: after vaccination, the MA(A) ELISA, like the CFT, was unable to differentiate infected cattle from those recently vaccinated with S19. After challenge the MA(A) ELISA gave results comparable with the CFT for those cattle with aberrant reactions. For the non-infected cattle there was a similar number of weeks after challenge when both tests were negative. It is suggested that the main advantage of the MA(A) ELISA when compared with the CFT lies in its relatively simple test procedure.     

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.     

Levamisole potentiation of antigen specific lymphocyte blastogenic response in Brucella abortus exposed but nonresponsive cattle

Incubation of Brucella abortus (field strain) infected and strain 19 vaccinated bovine peripheral blood lymphocytes with B. abortus antigen and levamisole caused a consistently significant increase in [3H] thymidine uptake when compared to cultures without levamisole. Levamisole did not potentiate B. abortus-induced blastogenic response of lymphocytes from non-exposed cattle. A dose response study showed that 10 micrograms/culture induced maximum potentiation of B. abortus-induced lymphocyte stimulation. Using the 10 micrograms/well concentration of levamisole, further studies were conducted to determine the net potentiation of the blastogenic responses in lymphocytes from B. abortus (field strain) infected cattle. B. abortus strain 19 vaccinated but nonresponsive and non-exposed cattle. Levamisole significantly potentiated the B. abortus-induced lymphocyte blastogenesis in lymphocytes from unresponsive cattle.     

Comparison of four serologic tests for the detection of antibodies to bovine leukemia virus

Four tests for detection of antibodies to bovine leukemia virus (BLV) were compared. The sera that were tested came from cattle in naturally infected commercial dairy herds, cattle that were infected under experimental conditions, and cattle in an isolated BLV-free herd. The tests that were compared included a radioimmunoprecipitation assay (RIA) with p24 antigen, a RIA with glycoprotein (gp) antigen, an agar-gel immunodiffusion (AGID) test with gp antigen, and a virus-neutralization (VN) test that was based on inhibition of BLV-induced syncytia in cell culture. Results of the 4 serologic tests agreed for 96.8% of the sera from cattle in commercial herds. The gp RIA detected the greatest number of positive sera (188); it was followed in turn by the p24 RIA (187), the VN test (183), and the AGID test (176). The gpd RIA titers of the 12 sera that gave negative AGID results were 175 or less. In RIA, the percentage of precipitation of labeled antigen by positive sera was almost always higher with gp antigen than with p24 antigen. Satisfactory sensitivity in the p24 RIA required the acceptance of a low level of antigen precipitation, 15%, as a positive test. In the gp RIA, however, almost all positive sera precipitated at least 50% of the labeled antigen. Nonspecific precipitation of antigen in the RIA by sera from BLV-free cattle ranged from 4% to 10%. Examination of sequential serum samples from 17 experimentally infected cattle showed that BLV antibody was first detected 2 to 8 weeks after inoculation. In 9 cattle, seroconversion was detected simultaneously by all of the tests. Results from the other 8 cattle indicated that seroconversion could be detected first by p24 RIA, followed by the gp RIA and the VN test. The longest interval between RIA seroconversion and AGID seroconversion was 10 days. Monthly tests of sera from 10 laboratory cattle that were infected by contact exposure showed that 7 animals seroconverted in all tests at the same time. Two cattle were positive first in RIA, but the next month they were also positive in the VN and AGID tests. One animal was positive in the RIA and the VN test for 2 months before antibody was detected by AGID.     

Evaluation of three Brucella soluble antigens used in an indirect Elisa to discriminate S19 vaccinated from naturally infected cattle

An O-polysaccharide (O-chain) and a hot-water extracted polysaccharide (PS), both obtained from Brucella abortus 1119-3, and a B. melitensis 16M native hapten (NH) were evaluated by indirect enzyme linked immunosorbent assay (ELISA) on three groups of cattle sera. The sera tested were: (a) 75 sera from cows naturally infected with B. abortus; (b) 130 sera from non-infected and non-vaccinated cattle; and (c) 61 sera from non-infected heifers recently vaccinated with B. abortus Strain 19 (S19). Sensitivity (Se), specificity (Sp) and the capability to discriminate vaccinated cattle (ADV) were determined. Using PS antigen, Se was 100% and the Sp was 97.7%, while the highest Sp was obtained by using the O-chain (99.2% ). For the NH antigen, Se was 94.7% and the Sp was 90.0%. The ADV of the three antigens was approximately 85%. Statistical analysis showed significant differences between O-chain/PS and O-chain/NH antigens. The agreement among antigens determined by kappa coefficient was 0.899 for O-chain/PS, 0.845 for O-chain/NH and 0.795 for PS/NH.