Lymphocyte proliferation in response to immunodominant antigens of Brucella abortus 2308 and RB51 in strain 2308-infected cattle.

Lymphocyte proliferation in response to proteins from the Brucella abortus strain 2308 (S2308) and the lipopolysaccharide (LPS) O-antigen-deficient mutant of S2308, strain RB51 (SRB51), was measured in S2308-infected cattle following abortion. Supramammary and superficial cervical lymph node lymphocytes from infected cattle proliferated most when incubated with 27- to 18-kDa proteins of S2308 or SRB51. Proteins of SRB51, which contained no LPS O antigens, induced lymphocyte proliferation similar to that induced by S2308 proteins, which contained LPS O antigens. These results indicate that 27- to 18-kDa proteins, but not LPS O antigens, of S2308 and SRB51 are immunodominant in S2308-infected cattle as assessed by lymphocyte proliferation assays.     

Lymphocyte proliferation in response to Brucella abortus 2308 or RB51 antigens in mice infected with strain 2308, RB51, or 19.

Lymphocyte proliferation to 22 protein fractions (106 to 18 kDa) of Brucella abortus 2308 or the lipopolysaccharide O-antigen-deficient mutant of 2308, strain RB51, was measured for 20 weeks after infection of mice with strain 2308, RB51, or 19. Throughout the 20-week study, the 22 protein fractions of 2308 and RB51 induced a similar pattern of proliferation when they were incubated with lymphocytes from the infected mice. In addition, during the 20 weeks, lymphocytes from all groups of infected mice exhibited the highest proliferation when the lymphocytes were incubated with 18-kDa or smaller proteins from either 2308 or RB51. Lymphocytes obtained from mice at 6 weeks after infection with strain RB51 or 19 exhibited similar proliferation to the 18-kDa proteins of S2308 or SRB51. Lymphocytes from strain 2308-infected mice did not proliferate to these proteins until 10 weeks after infection, and the responses were similar to those in strain RB51-infected mice but lower than those in strain 19-infected mice. Lymphocytes obtained from mice at 20 weeks after infection with strain 19 or 2308 proliferated to most of the 22 fractions of 2308 or RB51, which contained 106- to 18-kDa proteins. However, lymphocytes obtained from strain RB51-infected mice at 20 weeks did not proliferate to any of these fractions. These results indicate that mice infected with RB51 have less-persistent lymphocyte proliferative responses to 2308 proteins than do mice infected with 2308 or 19. In addition, all 2308 proteins that stimulate lymphocyte proliferation appear to be present in RB51.     

Antibody responses to Brucella abortus 2308 in cattle vaccinated with B. abortus RB51.

Cattle vaccinated with Brucella abortus rough strain RB51 (SRB51) produced small amounts of serum immunoglobulin G (IgG) but no IgM antibody to smooth strain 2308 (S2308) bacteria and produced no IgG or IgM antibody to S2308 lipopolysaccharide (LPS). Western immunoblot analysis revealed that antiserum from SRB51-vaccinated cattle contained IgG antibody that reacted with S2308 proteins of 84 to <20 kDa. However, antiserum from the vaccinated cattle did not contain agglutinating B. abortus antibody in the tube agglutination test for brucellosis. These results suggest that SRB51-vaccinated cattle produced no antibody to S2308 LPS, although they did produce nonagglutinating IgG antibody that reacted with S2308 bacteria and bacterial proteins of 84 to <20 kDa.     

Comparison of spleen cell proliferation in response to Brucella abortus 2308 lipopolysaccharide or proteins in mice vaccinated with strain 19 or RB51.

Mice vaccinated with Brucella abortus 19 (S19) or RB51 (SRB51) had spleen cells which proliferated in response to proteins of 32, 27, 18, and < 18 kDa but not in response to proteins of 106, 80, and 49 kDa from B. abortus 2308 (S2308) following vaccination and challenge infection with S2308. Spleen cells from mice vaccinated with S19 but not with SRB51 had increased proliferation in response to S2308 lipopolysaccharide (LPS) following challenge infection with S2308. We previously reported that mice vaccinated with S19 or SRB51, which were analyzed in the current study, have increased resistance to infection with S2308 and that only mice vaccinated with S19 produce antibody to S2308 LPS (M. Stevens, S. Olsen, G. Pugh, Jr., and D. Brees, Infect. Immun. 63:264-270, 1995). The results from our current and previous studies support the contention that vaccination of mice with S19 or SRB51 induces protection from infection with S2308 by cell-mediated immune responses to the same immunodominant (32, 27, 18, and < 18 kDa) protein antigens of S2308. In addition, the absence of S2308 LPS-responsive spleen cells and antibody to S2308 LPS in mice vaccinated with SRB51 suggests that immune responses to LPS have no role in SRB51-induced protective immunity.     

Mouse Cytokine Profiles Associated with Brucella abortus RB51 Vaccination or B. abortus 2308 Infection

This study indicated that mice immunized with Brucella abortus RB51 bacteria and subsequently challenged with B. abortus 2308 were protected from reinfection. After vaccination, both Th1 and Th2 cytokine patterns were observed. Of those, the early production of gamma interferon seems to have the prominent role in inducing an immunologically based protection.     

Immune responses and protection against experimental Brucella suis biovar 1 challenge in nonvaccinated or B. abortus strain RB51-vaccinated cattle

Twenty Hereford heifers approximately 9 months of age were vaccinated with saline (control) or 2 × 10(10) CFU of the Brucella abortus strain RB51 (RB51) vaccine. Immunologic responses after inoculation demonstrated significantly greater (P < 0.05) antibody and proliferative responses to RB51 antigens in cattle vaccinated with RB51 than in the controls. Pregnant cattle received a conjunctival challenge at approximately 6 months of gestation with 10(7) CFU of B. suis bv. 1 strains isolated from naturally infected cattle. The fluorescence polarization assay and the buffered acid plate agglutination test had the highest sensitivities in detecting B. suis-infected cattle between 2 and 12 weeks after experimental infection. Serologic responses and lymphocyte proliferative responses to B. suis antigens did not differ between control and RB51 vaccinees after experimental infection. No abortions occurred in cattle in either treatment group after challenge, although there appeared to be an increased incidence of retained placenta after parturition in both the control and the RB51 vaccination treatment groups. Our data suggest that the mammary gland is a preferred site for B. suis localization in cattle. Vaccination with RB51 did not reduce B. suis infection rates in maternal or fetal tissues. In conclusion, although B. suis is unlikely to cause abortions and fetal losses in cattle, our data suggest that RB51 vaccination will not protect cattle against B. suis infection after exposure.     

Immune and pathologic responses in mice infected with Brucella abortus 19, RB51, or 2308.

Immune and pathologic responses were measured for 20 weeks after infection of mice with Brucella abortus 19, RB51, or 2308. Live bacteria and bacterial antigens of 19 and RB51 persisted in spleens for 10 and 4 weeks after infection, respectively, whereas 2308 bacteria and bacterial antigens persisted for at least 20 weeks. Small germinal centers and profound lymphoid depletion occurred in spleens of mice during the first 4 weeks of infection with strain 19 or 2308; however, mice infected with strain RB51 had much larger germinal centers but no lymphoid depletion. At 4 weeks, only spleen cells from RB51-infected mice proliferated when incubated with 2308 bacteria. Large germinal centers in the spleen and spleen cell proliferative responses to 2308 did not appear in strain 19-infected mice until 6 weeks or in strain 2308-infected mice until 10 weeks. Similar proliferative responses to 2308 occurred in mice infected with strain 19 or RB51 at 6 weeks and in mice infected with strain 19, RB51, or 2308 at 10 weeks. However, at 20 weeks, spleen cell proliferative responses to 2308 occurred in mice infected with strain 19 or 2308 but not in mice infected with strain RB51. Mice infected with strain RB51 had lower and less persistent antibody titers to 2308 than did mice infected with strain 19 or 2308. Collectively, these results indicate that RB51-infected mice have less persistent immune responses to 2308 than do mice infected with 19 or 2308. The shorter duration of the responses probably resulted because RB51 is considerably less pathogenic and is cleared more rapidly from mice than are 19 and 2308.     

Brucella abortus RB51 induces protection in mice orally infected with the virulent strain B. abortus 2308

Brucellae are gram-negative, facultative intracellular bacteria which are one of the most common causes of abortion in animals. In addition, they are the source of a severe zoonosis. In this trial, we evaluated the effect of oral inoculation of Brucella abortus RB51 in mice against a challenge infection with B. abortus 2308. First, we showed that a gastric acid neutralization prior to the oral inoculation contributed to a more homogeneous and consistent infection with both vaccine strain B. abortus RB51 and virulent strain B. abortus 2308. Successively, we assessed the clearance and the immune response following an oral infection with B. abortus RB51. Oral inoculation gave a mild infection which was cleared 42 days after infection, and it induced a delayed humoral and cell-mediated immune response. Finally, we immunized mice by oral inoculation with B. abortus RB51, and we challenged them with the virulent strain B. abortus 2308 by an oral or intraperitoneal route 42 days after vaccination. Oral inoculation of B. abortus RB51 was able to give protection to mice infected with the virulent strain B. abortus 2308 by the oral route but not to mice infected intraperitoneally. Our results indicate that oral inoculation of mice with B. abortus RB51 is able to give a protective immunity against an oral infection with virulent strains, and this protection seems to rely on an immune response at the mucosal level.     

Serologic responses in diagnostic tests for brucellosis in cattle vaccinated with Brucella abortus 19 or RB51.

Serologic responses in the particle concentration fluorescence immunoassay and the card, complement fixation, and tube agglutination tests were measured for 10 weeks after vaccination of cattle with either Brucella abortus 19 or the lipopolysaccharide O-antigen-deficient mutant, strain RB51. The responses of strain 19-vaccinated cattle were positive, whereas those of strain RB51-vaccinated cattle were negative, in all of the tests. These results indicate that cattle vaccinated with strain RB51 fail to produce antibodies that can be detected by conventional serologic tests that are used to diagnose bovine brucellosis.     

Complement fixation test to assess humoral immunity in cattle and sheep vaccinated with Brucella abortus RB51.

The live attenuated Brucella abortus strain RB51 is a rifampin-resistant, lipopolysaccharide (LPS) O-chain-deficient mutant of virulent B. abortus 2308. The reduced O-chain content in RB51 prevents this bacterium from inducing antibodies detectable by the conventional serologic tests for bovine brucellosis diagnosis that mainly identify antibodies to LPS. The absence of available serologic tests for RB51 also complicates the diagnosis of possible RB51 infections in humans exposed to this strain. The purpose of this study was to evaluate the suitability of a complement fixation (CF) test performed with the rough strain B. abortus RB51, previously deprived of anticomplementary activity, in detecting anti-B. abortus RB51 antibodies in cattle and sheep experimentally vaccinated with this strain. The results of this study showed that a CF test with RB51 as the antigen is able to specifically detect antibodies following RB51 vaccination in cattle and sheep. In addition, this method could be a useful tool for detecting B. abortus RB51 infection in humans.     

Brucella abortus RB51 and Hot Saline Extract from Brucella ovis as Antigens in a Complement Fixation Test Used To Detect Sheep Vaccinated with Brucella abortus RB51

The efficacy of Brucella abortus RB51 and hot saline extract (HSE) from Brucella ovis as antigens in complement fixation (CF) tests was comparatively evaluated in detecting immune responses of sheep vaccinated with B. abortus strain RB51. For this study, four 5-month-old sheep were vaccinated subcutaneously with 5 × 10⁹ CFU of RB51, and two sheep received saline. Serum samples collected at different times after vaccination were tested for the presence of antibodies to RB51 by a CF test with RB51 as antigen, previously deprived of anticomplementary activity, and with HSE antigen, which already used as the official antigen to detect B. ovis-infected sheep. The results showed that vaccinated sheep developed antibodies which reacted weakly against HSE antigen and these antibodies were detectable for 30 days after vaccination. However, antibodies to RB51 could be detected for a longer period after vaccination by using homologous RB51 antigen in CF tests. In fact, high titers were still present at 110 days postvaccination with RB51 antigen. Sera from sheep naturally infected with B. ovis also reacted to RB51 but gave lower titers than those detected by HSE antigen. As expected, all sera from RB51-vaccinated sheep remained negative when tested with standard S-type Brucella standard antigens.     

Brucella abortus RB51 and hot saline extract from Brucella ovis as antigens in a complement fixation test used To detect sheep vaccinated with Brucella abortus RB51

The efficacy of Brucella abortus RB51 and hot saline extract (HSE) from Brucella ovis as antigens in complement fixation (CF) tests was comparatively evaluated in detecting immune responses of sheep vaccinated with B. abortus strain RB51. For this study, four 5-month-old sheep were vaccinated subcutaneously with 5 x 10(9) CFU of RB51, and two sheep received saline. Serum samples collected at different times after vaccination were tested for the presence of antibodies to RB51 by a CF test with RB51 as antigen, previously deprived of anticomplementary activity, and with HSE antigen, which already used as the official antigen to detect B. ovis-infected sheep. The results showed that vaccinated sheep developed antibodies which reacted weakly against HSE antigen and these antibodies were detectable for 30 days after vaccination. However, antibodies to RB51 could be detected for a longer period after vaccination by using homologous RB51 antigen in CF tests. In fact, high titers were still present at 110 days postvaccination with RB51 antigen. Sera from sheep naturally infected with B. ovis also reacted to RB51 but gave lower titers than those detected by HSE antigen. As expected, all sera from RB51-vaccinated sheep remained negative when tested with standard S-type Brucella standard antigens.     

Comparison of immune responses and resistance to brucellosis in mice vaccinated with Brucella abortus 19 or RB51.

Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following vaccination with B. abortus 19 (S19) or the lipopolysaccharide (LPS) O-antigen-deficient mutant, strain RB51 (SRB51). Live bacteria persisted for 8 weeks in spleens of mice vaccinated with 5 x 10(6) or 5 x 10(8) CFU of SRB51, whereas bacteria persisted for 12 weeks in mice vaccinated with 5 x 10(6) CFU of S19. Mice vaccinated with 5 x 10(6) or 5 x 10(8) CFU of SRB51 had increased resistance to infection with S2308 at 12, 16, and 20 weeks after vaccination, but the resistance was lower than that induced by vaccinating mice with 5 x 10(6) CFU of S19. Spleen cells obtained from mice vaccinated with S19 or SRB51 generally exhibited similar proliferative responses to S2308 bacteria or bacterial proteins (106 to 18 kDa) following challenge of mice with S2308 at 12, 16, or 20 weeks after vaccination. Mice vaccinated with S19 had antibody to S2308 bacteria and S2308 smooth LPS at 4, 8, and 12 weeks after vaccination. In contrast, mice vaccinated with either dose of SRB51 did not produce antibody to S2308 smooth LPS. In addition, only mice vaccinated with the highest dose of SRB51 (5 x 10(8) CFU) had antibody responses to S2308 bacteria, although the responses were lower and less persistent than those in mice vaccinated with S19. Collectively, these results indicate that SRB51-vaccinated mice have similar cell-mediated immune responses to S2308 but lower resistance to infection with S2308 compared with S19-vaccinated mice. The lower resistance in SRB51-vaccinated mice probably resulted from a combination of rapid clearance of SRB51 and an absence of antibodies to S2308 LPS.     

Coombs antiglobulin test using Brucella abortus 99 as antigen to detect incomplete antibodies induced by B abortus RB51 vaccine in cattle

This study showed that vaccination of cattle with Brucella abortus rough strain RB51 induces incomplete antibodies that can be detectable by a Coombs antiglobulin test using the B. abortus 99 smooth strain.     

Immune responses of elk to initial and booster vaccinations with Brucella abortus strain RB51 or 19

Previous studies have suggested that currently available brucellosis vaccines induce poor or no protection in elk (Cervus elaphus nelsoni). In this study, we characterized the immunologic responses of elk after initial or booster vaccination with Brucella abortus strains RB51 (SRB51) and 19 (S19). Elk were vaccinated with saline or 10(10) CFU of SRB51 or S19 (n=seven animals/treatment) and booster vaccinated with a similar dosage of the autologous vaccine at 65 weeks. Compared to nonvaccinates, elk vaccinated with SRB51 or S19 had greater (P<0.05) antibody responses to SRB51 or S19 after initial vaccination and after booster vaccination. Compared to nonvaccinated elk, greater (P<0.05) proliferative responses to autologous antigen after initial vaccination occurred at only a few sample times in SRB51 (6, 14, and 22 weeks) and S19 (22 weeks) treatment groups. In general, proliferative responses of vaccinates to nonautologous antigens did not differ (P>0.05) from the responses of nonvaccinated elk. Gamma interferon production in response to autologous or nonautologous Brucella antigens did not differ (P>0.05) between controls and vaccinates after booster vaccination. Flow cytometric techniques suggested that proliferation occurred more frequently in immunoglobulin M-positive cells, with differences between vaccination and control treatments in CD4+ and CD8+ subset proliferation detected only at 22 weeks after initial vaccination. After booster vaccination, one technique ([3H]thymidine incorporation) suggested that proliferative responses to SRB51 antigen, but not S19 antigen, were greater (P<0.05) in vaccinates compared to the responses of nonvaccinates. However, in general, flow cytometric and other techniques failed to detect significant anamnestic responses to autologous or nonautologous Brucella antigens in S19 or SRB51 vaccinates after booster vaccination. Although some cellular immune responses were detected after initial or booster vaccination of elk with SRB51 or S19, our data suggest that responses tend to be transient and much less robust than previously reported in SRB51-vaccinated cattle (Bos taurus) or bison (Bison bison). These data may explain why the vaccination of elk with S19 and SRB51 induces poor protection against brucellosis.     

Immune responses and resistance to brucellosis in mice vaccinated orally with Brucella abortus RB51.

Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following oral or intraperitoneal (i.p.) vaccination with strain RB51 (SRB51). Bacteria persisted in the parotid lymph node for 4 weeks following oral vaccination of mice with 5 x 10(8) or 5 x 10(6) CFU of SRB51. Bacteria did not appear in the spleen during 12 weeks after oral vaccination, whereas they did appear in the spleen for 8 weeks following i.p. vaccination of mice with SRB51 (5 x 10(8) or 5 x 10(6) CFU). Increased resistance to S2308 infection occurred at 12 to 20 weeks in mice vaccinated i.p. with SRB51 (5 x 10(8) or 5 x 10(6) CFU) but occurred at 12 weeks only in mice vaccinated orally with SRB51 (5 x 10(8) CFU). Oral SRB51 vaccination induced lower levels of antibodies to the surface antigens of intact SRB51 bacteria than did i.p. vaccination. However, neither route of vaccination induced anamnestic antibody responses to the surface antigens of intact S2308 bacteria after challenge infection of the vaccinated mice with S2308. Mice vaccinated orally with SRB51 and challenged with S2308 at 12 to 20 weeks had lower and less persistent spleen cell proliferation and production of gamma interferon in response to S2308 and certain immunodominant S2308 proteins (32 to < or = 18 kDa) than did mice vaccinated i.p. with SRB51. However, mice vaccinated orally or i.p. with SRB51 and challenged with S2308 had similar spleen cell tumor necrosis factor alpha production. These results indicate that oral vaccination of mice with SRB51 was effective in inducing protective immunity to S2308 infection, although the immunity was lower and less persistent than that induced by i.p. vaccination. The lower protective immunity induced by oral vaccination may have resulted from lower and less persistent cell-mediated immunity and gamma interferon production in response to S2308 and S2308 proteins.     

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

Immune Responses and Safety after Dart or Booster Vaccination of Bison with Brucella abortus Strain RB51

One alternative for management of brucellosis in Yellowstone National Park bison (Bison bison) is vaccination of calves and yearlings. Although Brucella abortus strain RB51 vaccination protects bison against experimental challenge, the effect of booster vaccinations was unknown. This study characterized immunologic responses after dart or booster vaccination of bison with Brucella abortus strain RB51. In two studies, 8- to 10-month-old female bison were inoculated with saline (n = 14), hand vaccinated with 1.1 × 10¹⁰ to 2.0 × 10¹⁰ CFU of RB51 (n = 21), or dart vaccinated with 1.8 × 10¹⁰ CFU of RB51 (n = 7). A subgroup of hand vaccinates in study 1 was randomly selected for booster vaccination 15 months later with 2.2 × 10¹⁰ CFU of RB51. Compared to single vaccinates, booster-vaccinated bison had greater serologic responses to RB51. However, there was a trend for antigen-specific proliferative responses of peripheral blood mononuclear cells (PBMC) from booster vaccinates to be reduced compared to responses of PBMC from single vaccinates. PBMC from booster vaccinates tended to have greater gamma interferon (IFN-γ) production than those from single vaccinates. In general, dart vaccination with RB51 induced immunologic responses similar to those of hand vaccination. All vaccinates (single hand, dart, or booster) demonstrated greater (P < 0.05) immunologic responses at various times after vaccination than nonvaccinated bison. Booster vaccination with RB51 in early gestation did not induce abortion or fetal infection. Our data suggest that booster vaccination does not induce strong anamnestic responses. However, phenotypic data on resistance to experimental challenge are required to fully assess the effect of booster vaccination on protective immunity.     

Induction of specific cytotoxic lymphocytes in mice vaccinated with Brucella abortus RB51

A safe, more sensitive, nonradioactive, neutral red uptake assay was adopted to replace the traditional 51Cr release assay for detection of Brucella-specific cytotoxic T lymphocyte (CTL) activity. Our studies indicated that Brucella abortus strain RB51 vaccination of mice induced specific CTLs against both strain RB51- and strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. The antigen-specific cytotoxic activity was exerted by T lymphocytes but not by NK cells. CD3+ CD4+ T cells secreted the highest level of gamma interferon (IFN-gamma) and were able to exert a low but significant level of specific lysis of Brucella-infected macrophages. They also exerted a low level of nonspecific lysis of noninfected macrophages. In contrast, CD3+ CD8+ T cells secreted low levels of IFN-gamma but demonstrated high levels of specific lysis of Brucella-infected macrophages with no nonspecific lysis. These findings indicate that B. abortus strain RB51 vaccination of mice induces specific CTLs and suggest that CD3+ CD4+ and CD3+ CD8+ T cells play a synergistic role in the anti-Brucella activity.     

Brucella abortus 1119-3 O-chain polysaccharide to differentiate sera from B. abortus S-19-vaccinated and field-strain-infected cattle by agar gel immunodiffusion.

Purified Brucella abortus 1119-3 and Brucella melitensis 16M lipopolysaccharide O-chain polysaccharides were not precipitated in agar gel immunodiffusion by any of 24 sera from vaccinated cattle but were precipitated by 18 of 24 sera from infected cattle. This difference can be used to differentiate sera of cattle vaccinated with B. abortus S-19 from sera of some field-strain-infected cattle.