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.     

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.     

Field Validation of the Use of RB51 as Antigen in a Complement Fixation Test To Identify Calves Vaccinated with Brucella abortus RB51

In order to confirm the efficiency of an experimental RB51-based complement fixation (CF) test in identifying cattle vaccinated with Brucella abortus strain RB51, 831 sera from 110 vaccinated and 48 unvaccinated Hereford heifers of Iowa, collected for studies conducted in different years, were sent to Italy without coding to be tested in a CF test using RB51 as antigen. Most of the calves, aged from 3 to 10 months, were vaccinated subcutaneously with the recommended dosage of 10¹⁰ CFU of RB51 commercial vaccine, while only six calves received 10⁹ CFU of the same vaccine. Serum samples for serologic testing, collected until 16 postinoculation weeks (PIW), were also tested by routine surveillance tests for brucellosis such as rose bengal plate and CF tests performed with B. abortus smooth strain 99 as control antigen. RB51 CF test results obtained by testing sera from cattle vaccinated in 1999 indicate that the sensitivity of the reaction is 97% at 2 to 3 PIW and 90% until 8 PIW and decreases to 65% at 12 PIW, the specificity remaining at 100%. Collectively, the results of this study confirm that serologic standard tests fail to detect antibodies to RB51 while the RB51-based CF test is able to monitor antibody responses to RB51 until 15 to 16 PIW with a specificity of 100%. In addition, unlike the RB51-based dot blot assay, which is the only test currently used to monitor antibody responses to RB51, the CF test also detected specific responses following vaccination with 10⁹ CFU of RB51, although seroconversion was only 50% at 8 PIW. In conclusion, because of high specificity and sensitivity, the CF test described here can be used to efficaciously monitor serologic responses following RB51 vaccination in cattle and could also be employed to detect RB51 infection in humans exposed to this strain.     

Rough Lipopolysaccharide of Brucella abortus RB51 as a Common Antigen for Serological Detection of B. ovis,B. canis,and B. abortus RB51 Exposure Using Indirect Enzyme Immunoassay and Fluorescence Polarization Assay

Abstract

Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.     

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.     

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.     

Cloning and Sequencing of yajC and secD Homologs of Brucella abortus and Demonstration of Immune Responses to YajC in Mice Vaccinated with B. abortus RB51

To identify Brucella antigens that are potentially involved in stimulating a protective cell-mediated immune response, a gene library of Brucella abortus 2308 was screened for the expression of antigens reacting with immunoglobulin G2a antibodies from BALB/c mice vaccinated with B. abortus RB51. One selected positive clone (clone MCB68) contained an insert of 2.6 kb; nucleotide sequence analysis of this insert revealed two open reading frames (ORFs). The deduced amino acid sequences of the first and second ORFs had significant similarities with the YajC and SecD proteins, respectively, of several bacterial species. Both the YajC and SecD proteins were expressed in Escherichia coli as fusion proteins with maltose binding protein (MBP). In Western blots, sera from mice vaccinated with B. abortus RB51 recognized YajC but not SecD. Further Western blot analysis with purified recombinant YajC protein indicated that mice inoculated with B. abortus 19 or 2308 or B. melitensis RM1 also produced antibodies to YajC. In response to in vitro stimulation with recombinant MBP-YajC fusion protein, splenocytes from mice vaccinated with B. abortus RB51 were able to proliferate and produce gamma interferon but not interleukin-4. This study demonstrates, for the first time, the involvement of YajC protein in an immune response to an infectious agent.     

Immune Responses of Bison and Efficacy after Booster Vaccination with Brucella abortus Strain RB51

Thirty-one bison heifers were randomly assigned to receive saline or a single vaccination with 10¹⁰ CFU of Brucella abortus strain RB51. Some vaccinated bison were randomly selected for booster vaccination with RB51 at 11 months after the initial vaccination. Mean antibody responses to RB51 were greater (P < 0.05) in vaccinated bison after initial and booster vaccination than in nonvaccinated bison. The proliferative responses by peripheral blood mononuclear cells (PBMC) from the vaccinated bison were greater (P < 0.05) than those in the nonvaccinated bison at 16 and 24 weeks after the initial vaccination but not after the booster vaccination. The relative gene expression of gamma interferon (IFN-γ) was increased (P < 0.05) in the RB51-vaccinated bison at 8, 16, and 24 weeks after the initial vaccination and at 8 weeks after the booster vaccination. The vaccinated bison had greater (P < 0.05) in vitro production of IFN-γ at all sampling times, greater interleukin-1β (IL-1β) production in various samplings after the initial and booster vaccinations, and greater IL-6 production at one sampling time after the booster vaccination. Between 170 and 180 days of gestation, the bison were intraconjunctivally challenged with approximately 1 × 10⁷ CFU of B. abortus strain 2308. The incidences of abortion and infection were greater (P < 0.05) in the nonvaccinated bison after experimental challenge than in the bison receiving either vaccination treatment. Booster-vaccinated, but not single-vaccinated bison, had a reduced (P < 0.05) incidence of infection in fetal tissues and maternal tissues compared to that in the controls. Compared to the nonvaccinated bison, both vaccination treatments lowered the colonization (measured as the CFU/g of tissue) of Brucella organisms in all tissues, except in retropharyngeal and supramammary lymph nodes. Our study suggests that RB51 booster vaccination is an effective vaccination strategy for enhancing herd immunity against brucellosis in bison.     

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 Antigen-Specific Th1-Type Immune Responses by Gamma-Irradiated Recombinant Brucella abortus RB51

Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli β-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize β-galactosidase. A single intraperitoneal inoculation of mice with 10⁹ CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a β-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice.     

Field validation of the use of RB51 as antigen in a complement fixation test to identify calves vaccinated with Brucella abortus RB51

In order to confirm the efficiency of an experimental RB51-based complement fixation (CF) test in identifying cattle vaccinated with Brucella abortus strain RB51, 831 sera from 110 vaccinated and 48 unvaccinated Hereford heifers of Iowa, collected for studies conducted in different years, were sent to Italy without coding to be tested in a CF test using RB51 as antigen. Most of the calves, aged from 3 to 10 months, were vaccinated subcutaneously with the recommended dosage of 10(10) CFU of RB51 commercial vaccine, while only six calves received 10(9) CFU of the same vaccine. Serum samples for serologic testing, collected until 16 postinoculation weeks (PIW), were also tested by routine surveillance tests for brucellosis such as rose bengal plate and CF tests performed with B. abortus smooth strain 99 as control antigen. RB51 CF test results obtained by testing sera from cattle vaccinated in 1999 indicate that the sensitivity of the reaction is 97% at 2 to 3 PIW and 90% until 8 PIW and decreases to 65% at 12 PIW, the specificity remaining at 100%. Collectively, the results of this study confirm that serologic standard tests fail to detect antibodies to RB51 while the RB51-based CF test is able to monitor antibody responses to RB51 until 15 to 16 PIW with a specificity of 100%. In addition, unlike the RB51-based dot blot assay, which is the only test currently used to monitor antibody responses to RB51, the CF test also detected specific responses following vaccination with 10(9) CFU of RB51, although seroconversion was only 50% at 8 PIW. In conclusion, because of high specificity and sensitivity, the CF test described here can be used to efficaciously monitor serologic responses following RB51 vaccination in cattle and could also be employed to detect RB51 infection in humans exposed to this strain.     

Identification of an IS711 Element Interrupting the wboA Gene of Brucella abortus Vaccine Strain RB51 and a PCR Assay To Distinguish Strain RB51 from Other Brucella Species and Strains

Brucella abortus vaccine strain RB51 is a natural stable attenuated rough mutant derived from the virulent strain 2308. The genetic mutations that are responsible for the roughness and the attenuation of strain RB51 have not been identified until now. Also, except for an assay based on pulsed-field gel electrophoresis, no other simple method to differentiate strain RB51 from its parent strain 2308 is available. In the present study, we demonstrate that the wboA gene encoding a glycosyltransferase, an enzyme essential for the synthesis of O antigen, is disrupted by an IS711 element in B. abortus vaccine strain RB51. Exploiting this feature, we developed a PCR assay that distinguishes strain RB51 from all other Brucella species and strains tested.     

Efficacy of Dart or Booster Vaccination with Strain RB51 in Protecting Bison against Experimental Brucella abortus Challenge

This study characterized the efficacy of the Brucella abortus strain RB51 vaccine in bison when delivered by single intramuscular vaccination (hand RB51), by single pneumatic dart delivery (dart RB51), or as two vaccinations approximately 13 months apart (booster RB51) in comparison to control bison. All bison were challenged intraconjunctivally in midgestation with 10⁷ CFU of B. abortus strain 2308 (S2308). Bison were necropsied and sampled within 72 h of abortion or delivery of a live calf. Compared to nonvaccinated bison, bison in the booster RB51 treatment had a reduced (P < 0.05) incidence of abortion, uterine infection, or infection in maternal tissues other than the mammary gland at necropsy. Bison in single-vaccination treatment groups (hand RB51 and dart RB51) did not differ (P > 0.05) from the control group in the incidence of abortion or recovery of S2308 from uterine, mammary, fetal, or maternal tissues at necropsy. Compared to nonvaccinated animals, all RB51 vaccination groups had reduced (P < 0.05) mean colonization or incidence of infection in at least 2 of 4 target tissues, with the booster RB51 group having reduced (P < 0.05) colonization and incidence of infection in all target tissues. Our data suggest that booster vaccination of bison with RB51 enhances protective immunity against Brucella challenge compared to single vaccination with RB51 by hand or by pneumatic dart. Our study also suggests that an initial vaccination of calves followed by booster vaccination as yearlings should be an effective strategy for brucellosis control in bison.     

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 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 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.     

Overexpression of protective antigen as a novel approach to enhance vaccine efficacy of Brucella abortus strain RB51

Brucella abortus strain RB51 is an attenuated rough strain that is currently being used as the official live vaccine for bovine brucellosis in the United States and several other countries. We reasoned that overexpression of a protective antigen(s) of B. abortus in strain RB51 should enhance its vaccine efficacy. To test this hypothesis, we overexpressed Cu/Zn superoxide dismutase (SOD) protein of B. abortus in strain RB51. This was accomplished by transforming strain RB51 with a broad-host-range plasmid, pBBR1MCS, containing the sodC gene along with its promoter. Strain RB51 overexpressing SOD (RB51SOD) was tested in BALB/c mice for its ability to protect against challenge infection with virulent strain 2308. Mice vaccinated with RB51SOD, but not RB51, developed antibodies and cell-mediated immune responses to Cu/Zn SOD. Strain RB51SOD vaccinated mice developed significantly (P < 0.05) more resistance to challenge than those vaccinated with strain RB51 alone. The presence of the plasmid alone in strain RB51 did not alter its vaccine efficacy. Also, overexpression of SOD did not alter the attenuation characteristic of strain RB51.     

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.     

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.     

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.