Preliminary evidence for a diagnostic immunoglobulin G1 antibody response among culture-positive cows vaccinated with Brucella abortus strain 19 and challenge exposed with strain 2308

The sera of cows inoculated with Brucella abortus have a characteristically high titer of immunoglobulin (Ig) G1 antibodies to a soluble brucella antigen compared with sera of noninoculated vaccinated cattle. Concentrations of antigen-specific IgG1 were greater than 10-fold higher than those for IgG2, even though total IgG2 concentrations were higher than total IgG1 concentrations. Increases in IgG1 antibodies to Brucella abortus soluble antigen were detected shortly after vaccination in those cows from which strain 19 was isolated and by 28 weeks in cows from which strain 2308 was isolated. Increases in specific antibodies were not paralleled by increases in either total IgG1 or total IgG2 concentrations. Rather, there was a 15-fold to greater than 200-fold increase in specific activity, with up to 16% of the IgG1 specific for the brucella antigen used in the assay. Thus, measurement of changes in total IgG1 concentrations is not a reliable method to identify brucellosis-associated anti-Brucella abortus soluble antigen activity. Only one cow in a panel of 10 selected for detailed study showed a false-positive IgG1 titer, whereas some serologic assays showed as many as 4 or 5 false-positives. Results of the complement-fixation test, among the battery of serologic tests used for detection of brucellosis, best agreed with the occurrence of increased IgG1 antibody levels.     

Comparison of the results of card, rivanol, complement-fixation, and milk ring tests with the isolation rate of Brucella abortus from cattle

Specimens from 4,553 cows were examined by card, Rivanol, and complement-fixation (CF) tests and bacteriologic culture. A ring test was performed on milk from 1,003 of these cows. The isolation rate of Brucella abortus correlated directly with antibody titers, and the field strain predominated in adult-vaccinated cows when the Rivanol test titer was greater than + 100 and the CF test titer was greater than 4 + 40. The CF test had the best balance of sensitivity and specificity in adult-vaccinated cows. The false-negative rate for the Rivanol and CF tests was higher in nonadult-vaccinated cows.     

Detection of brucella antibodies of different immunoglobulin classes in cow milk by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assays were conducted on milk of cows from which Brucella abortus was isolated and that of noninfected controls. Horseradish peroxidase-labeled rabbit antibovine immunoglobulins IgG, IgG1, and IgA were used as conjugates. A heat-killed whole-cell suspension of B abortus strain 19 was used as the antigen. Differences in antibody profiles were observed in milk of cows from which B abortus was isolated and in milk of noninfected cows. Antibody profiles were similar in milk of cows infected with B abortus and that of cows from which B abortus strain 19 was isolated.     

VACCINATION OF PREGNANT COWS WITH LOW DOSES OF BRUCELLA ABORTUS STRAIN 19 VACCINE

Two groups, each of 9 Jersey cows, were vaccinated subcutaneously with reduced doses of Brucella abortus strain 19 vaccine (measured by the number of bacteria in the vaccine dose) early in their second pregnancy. Ten weeks later they were challenged, along with a similar group of non-vaccinated cows, by conjunctival instillation of a virulent strain of B. abortus biotype 1. Cows in Group 1 received 1/20th and those in Group 2 received 1/400th the recommended dose of strain 19. Marked reduction in the serological response to vaccination was seen only in Group 2. Four cows in Group 1 excreted strain 19 after parturition, one of them aborted and another calved prematurely with heavy infection of the placenta and foetus with strain 19 in both cases. Resistance to challenge was similar in both vaccinated groups, and higher than previously demonstrated after conventional calfhood vaccination with strain 19. It is concluded that pregnant cows can be effectively vaccinated by the subcutaneous administration of a dose of strain 19 vaccine containing approximately 3 x 10(8) organisms without undue interference with subsequent serological tests or inconvenience resulting from persistence of strain 19 infection.     

The role of the differential complement fixation test, using rough and smooth brucella antigens, in the anamnestic test

To assess the ability of the differential complement fixation test to distinguish vaccinal reactors from infected cattle, approximately 1,000 heifers were tested by the complement fixation test (CFT) using rough and smooth brucella antigens, before the injection of 45/20 vaccine and at 3 and 6 or 10 weeks after vaccination. Before vaccination 91.5% of heifers were negative to the rough antigen but 0.6% were positive with high titre (greater than or equal to 128). By 10 weeks after injection of 45/20 vaccine 97.6% of heifers were positive to the rough CF antigen, at greater than or equal to 8, a majority reaching greater than or equal to 128. Nineteen pre-vaccinal reactors to the standard CFT were killed and Brucella abortus was isolated from the tissues of 14. Twenty-six post-vaccinal reactors were killed and B. abortus was isolated from the tissues of 8. In the 22 B. abortus infected animals the differential CFT classified 9 correctly as infected, 5 incorrectly as vaccination reactions and 8 as inconclusive. The differential CF was ineffective in distinguishing titres resulting from vaccination with 45/20 vaccine from those due to infection.     

Experimental infection of dogs with Brucella abortus

Thirteen female dogs, which included eight principals that were fed approximately 4.4 X 10(10) colony forming units (cfu) of Brucella abortus strain 2308 and five sentinels that were housed with the principals, were examined for serologic responses, blood culture, tissue distribution of the organisms and pathologic lesions. Serum samples from each dog were tested on the day of exposure and on post exposure days 5, 7, 10, 14, 21, 28, 35, 42 and 49 for antibodies to B. abortus, using the brucellosis card (BC), standard tube agglutination (STA), 2-mercaptoethanol (ME) and rivanol (RIV) tests. Antibodies were detected in the principals by day 5 and increased through day 21. The STA test was the first to become positive, followed by the BC, ME and RIV tests. After 28 days, the serologic titers receded. From day 14 through day 42, all principals had greater than or equal to 1:50 STA titers. On day 49, seven principals had greater than or equal to 1:50 STA titers and one had a 1:25 STA titer. The sentinels were negative for all tests, except sentinel number 9 which had STA titers ranging from 1:25 to 1:50 on day 14 through day 35. Blood cultures that were obtained from each principal at intervals from one hour after exposure through 49 days were negative. Brucella abortus was isolated from various lymph nodes of the eight principals and from sentinel number 9, which was apparently infected by ingesting brucellae contaminated feces from the principals. Microscopic lesions were not observed in the culture-positive tissues examined.     

Brucella abortus-specific immunoglobulin in isotypes in serum and vaginal mucus from cattle vaccinated with strain 19 and challenge exposed with virulent strain 2308

The immunoglobulins (IgG1, IgG2, IgM, and IgA) of the Brucella-specific antibody response of 69 crossbred beef heifers were studied after Brucella abortus strain 19 vaccination and strain 2308 challenge exposure. The immunoglobulin isotype responses in serum and vaginal mucus were measured by use of fluorescent immunoassay. Serum antibody responses were detected also by 3 standard serologic tests (complement fixation [CF], Rivanol precipitation, and the CARD test] and 2 primary bindings assays that detect IgG antibodies. One month after vaccination, mean antibody titers for all immunoglobulin isotypes were higher for vaccinated cattle (n = 46) than for nonvaccinated controls (n = 23). After vaccination, IgA antibody responses in vaccinated cattle were only 2-fold higher than those for controls, whereas IgG1, IgG2, and IgM antibody responses were 3- to 90-fold greater than those for controls. Measurement of IgA antibody responses classified 21 of 39 vaccinates as seropositive after vaccination, whereas the other isotypes classified 28 or 34 cattle as seropositive. Three months after challenge exposure, the mean antibody responses for each isotype were higher in cattle that aborted or were culture positive than in cattle that did not abort and were culture negative. Although IgG1, IgG2, and IgM antibody titers were each of benefit in identifying B abortus-infected cattle, it did not appear that the magnitude of the antibody responses provided sufficient discrimination between S19-vaccinated cattle and S2308 challenge-exposed cattle. Serum IgA antibody responses were 10-fold higher after challenge exposure than after vaccination and may be a response to mucosal infection with the virulent organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Effect of dose of Brucella abortus strain 19 in yearling heifers on the relative risk of developing brucellosis from challenge exposure with strain 2308

Yearling heifers were given SC injections of 10(8) (n = 40), 10(9) (n = 44), or 10(10) (n = 44) colony-forming units of Brucella abortus strain 19 (S19). The proportion of heifers with positive serologic test results at 1 month following vaccination increased as the dose of S19 increased. These proportions decreased with time, and all heifers had negative card, rivanol, and complement fixation test results within 4 months. Positive ELISA results persisted beyond 4 months in all three S19 dose groups; however, all heifers were ELISA-negative within 9 months after vaccination. Comparable lymphocyte transformation activity was stimulated by S19 dose of 10(9) or 10(10) and approximately half of the heifers in both groups had a positive stimulation index at 9 months. Immunity of the pregnant heifers was challenged 9 months after vaccination with 10(7) B abortus strain 2308 as follows: diluent controls (n = 69); 10(8) B abortus S19 (n = 40); 10(9) B abortus S19 (n = 39); and 10(10) B abortus S19 (n = 39). Tissue specimens from heifers were obtained at parturition and necropsy for culturing of B abortus. The proportion of heifers that developed brucellosis, ie, had positive culture results, increased as gestation days at challenge exposure increased. The effect of gestational age was controlled in the analysis using logistic regression. The relative risk of brucellosis was reduced to 0.38, 0.15, and 0.06 for B abortus S19 doses of 10(8), 10(9), and 10(10), respectively, compared with diluent controls at 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Isolation of Brucella abortus and Brucella abortus, strain 19, from cattle.

Tissues from 104 cows in herd were examined for brucellae. Brucella abortus, strain 19, was isolated from 22 cows, a field strain of B abortus, biotype 1, was isolated from 9 cows, and both strains were isolated from 2 cows.     

Identification of a virulence factor for Brucella abortus infection in BALB/c mice

Immunogenic or pathogenic factors of recombinant proteins (rBCSP20, rBCSP-31, and rBCSP45 of Brucella abortus strain 19) for mice were compared with factors of a proteinase K-treated lipopolysaccharide extracted from B abortus strain 2308. Mice were vaccinated with 4 products, using different inoculation schedules and were challenge exposed with a virulent culture of B abortus strain 2308. Blood samples were collected 2 weeks after vaccination and at necropsy and sera were obtained. Spleens were cultured for B abortus at necropsy (3 to 4 weeks after challenge exposure). Mice given proteinase K-treated lipopolysaccharide alone or in conjunction with rBCSP20 or rBCSP45 proteins were protected, but mice given rBCSP31 on the same day as challenge exposure were not. Vaccination with recombinant proteins alone neither provide protection nor significantly (P greater than 0.05) increase the pathogenic effect of the challenge-exposure culture. Seemingly, rBCSP31 might be a virulence factor of B abortus.     

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.     

Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of Brucella abortus

The outer membrane-peptidoglycan complex (OM-PG) from rough strains of Brucella abortus was tested for its ability to induce lymphocyte responsiveness in cattle. Six groups of heifers were immunized with varying doses and administration schedules of rough OM-PG and assayed for responsiveness of their lymphocytes in proliferation assays in vitro. All OM-PG preparations were emulsified in a commercial adjuvant for administration. Two other groups of heifers were immunized with strain 19 vaccine or adjuvant alone. Three groups of heifers received two inoculations of OM-PG antigens from a naturally-occurring rough strain at a 57-day interval. The doses of OM-PG given in these three groups were 400 micrograms, 1200 micrograms, and 4000 micrograms at each inoculation. The frequency of cows that responded in lymphocyte proliferation assays increased with the dose of OM-PG given. Two groups received single inoculations of OM-PG, either 2400 micrograms or 8000 micrograms. Although there were responsive cows in these immunization groups, their frequency was lower than in the groups receiving the same total dose in two inoculations. A sixth group of cows was inoculated with OM-PG from a rough transposon mutant of B. abortus, and the frequency of responsive cows in this immunization group was comparable to that of responsive cows immunized with the same dose of OM-PG from the spontaneous rough mutant. In comparisons of cows inoculated with strain 19 to those inoculated with OM-PG preparations, differences were observed in the relative responsiveness of their lymphocytes to whole cells and OM-PG in the in vitro lymphocyte proliferation assays. These differences suggested that lymphocytes stimulated by strain 19 vaccination have different specificities than those stimulated by immunization with OM-PG of rough mutant strains of B. abortus.     

Antibody isotype response in adult cattle vaccinated with Brucella abortus S19

In a chronological study of sera collected from eight adult cattle vaccinated with 3 X 10(-10) cfu of Brucella abortus S19, antibody of each of the four major isotypes was measured by indirect enzyme immunoassay (ELISA) and by direct and modified complement fixation tests (CFT). Six of the cattle gave antibody responses to the vaccine strain that commenced between days 5 and 8 for all the isotypes in the ELISA, peaked by 1 to 4 months and then declined to low levels by 10 months. Direct CFT and modified CFT titers were measurable by 7 or 8 days post-vaccination, and peaked by 1 month; direct CFT titers disappeared by 5 months while the modified CFT titers lingered for 10 months. Two animals gave cyclical direct CFT and modified CFT antibody responses, a cyclical IgG1 response, a low IgG2 and an elevated IgA response. The amplitude of the cycles was uniform over three cycles while the wavelength increased with time. A year post-vaccination, B. abortus S19 was isolated twice from milk from one of the animals (no attempt was made to culture B. abortus from the other). Sera from B. abortus naturally infected cattle were analysed for comparison.     

Efficacy of calfhood vaccination with Brucella abortus strain RB51 in protecting bison against brucellosis

In the studies reported here, protection induced by calfhood vaccination of bison with 1.2-6.1 x 10(10)CFU of Brucella abortus strain RB51 (SRB51) against a virulent strain of B. abortus was evaluated. Non-vaccinated and SRB51-vaccinated bison were intraconjunctivally challenged during midgestation with 3 x 10(7)CFU of virulent B. abortus strain 2308 (S2308). Maternal and fetal tissues were obtained within 24hour after abortion or parturition. Incidence of abortion was greater (P<0.05) in non-vaccinated as compared to SRB51-vaccinated bison (62% and 15%, respectively), with abortions occurring between 5 and 8 weeks after experimental challenge. Calves from bison vaccinated with SRB51 had a reduced (P<0.05) prevalence of fetal infection with S2308 as compared to calves from non-vaccinated bison (19% and 62%, respectively). Although the ability to recover the 2308 challenge strain from maternal tissues did not differ (P>0.05) between nonvaccinates and vaccinates (100% and 78%, respectively), calfhood vaccination with SRB51 reduced (P<0.05) recovery of S2308 from uterine or mammary gland tissues. In bison which did not abort, S2308 was routinely recovered in low numbers from maternal lymphatic tissues; particularly the parotid, bronchial, supramammary, and mandibular lymph nodes. The RB51 vaccine strain was not recovered at any time from maternal or fetal samples obtained at necropsy. Histological lesions associated with Brucella-induced abortions were suppurative placentitis, fetal broncho-interstitial pneumonia, and fetal histiocytic splenitis. The results of our studies suggest that calfhood vaccination of bison with SRB51 is efficacious in protecting against intramammary, intrauterine, and fetal infection following exposure to a virulent strain of B. abortus during pregnancy. As brucellosis is transmitted horizontally through fluids associated with the birth or abortion of an infected fetus, or vertically to the calf through the ingestion of milk containing B. abortus, our data suggest that calfhood vaccination with SRB51 will be beneficial in preventing transmission of brucellosis in bison.     

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.     

Vesicular stomatitis virus (New Jersey strain) infection in two California dairy herds: an epidemiologic study

The course of vesicular stomatitis in cattle was investigated in 2 dairy herds (A and B) located in the southern San Joaquin Valley of California. Cattle were examined and specimens were obtained for virus isolation and for serologic survey for one year after an epizootic in December 1982. All 33 lactating cows selected for study had oral lesions, but only 19 (58%) were drooling or frothing around the mouth. Lesions on feet and teats were not observed. The healing time (longer than has been reported previously) for oral lesions ranged from 34 to 59 days. The mean serum neutralizing antibody titer for all cows tested in both herds 21 days after clinical signs were first observed was greater than 1:512. The mean titer decreased in the first 11 months after the epizootic, but remained greater than 1:128, and then increased during December 1983. Vesicular stomatitis virus/New Jersey strain was not isolated from 239 blood samples, 235 swab specimens of oral cavities, 38 swab specimens of oral epithelium, 206 urine specimens, or 232 fecal specimens collected from cows; however, it was isolated from tongue epithelium of 3 cows at 1, 4, and 21 days after signs of frothing were first noticed. For 20 lactating cows brought into dairy A during the epizootic, a mean time of 8.9 days elapsed between time of entry and appearance of clinical signs of vesicular stomatitis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of various treatment regimens, using liposomal streptomycin in cows with brucellosis

Thirty cows naturally infected with Brucella abortus were treated by various routes, using free or liposomal streptomycin or a combination of liposomal streptomycin and a long-acting oxytetracycline preparation. Of 21 cows treated with liposomal streptomycin alone, 3 (14%) were culture negative and 3 had 10 or fewer bacterial colonies isolated from tissues obtained at necropsy. Thirteen (62%) cows continued to shed organisms in udder secretions and were considered treatment failures. Of 9 cows that were given a combination of liposomal streptomycin and long-acting oxytetracycline, 5 (56%) were cured, 3 had 10 or fewer colonies on culture plates of tissue after necropsy and only 1 continued to shed B abortus in udder secretions after treatment. Eleven cows were given streptomycin liposomes by intramammary infusion with or without IM administration of long-acting oxytetracycline. The most effective regimen consisted of 2 intramammary infusions of streptomycin liposomes and 2 doses of oxytetracycline administered IM. Of 5 cows treated thusly, 2 were cured and all others had fewer than 10 B abortus colonies isolated from tissues obtained at necropsy.