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Vaccine Comparison

B. abortus strain 19 B. abortus vaccine strain RB51
Vaccine Information Vaccine Information
  • Vaccine Ontology ID: VO_0000022
  • Type: Attenuated live vaccine
  • Status: Licensed
  • Location Licensed: USA; European countries
  • Host Species for Licensed Use: Cattle
  • Antigen: Whole Brucella organism
  • Preparation: Strain 19, first described in 1930, is a laboratory-derived strain attenuated by an unknown process. It was originally isolated from bovine milk as a virulent strain in 1923, but became attenuated after storage at room temperature for over a year. Strain 19 is able to induce protective immunity in cattle (Schurig et al., 2002).
  • Virulence: Although effective, strain 19 vaccine has a tropism for the placenta and causes abortion when given to pregnant cows , is infectious for humans , and causes serologic responses in calves that are the same as those in cattle infected with natural field strains (Cheville, 2000).
  • Description: Strain 19 induces reasonable protection against B. abortus, but at the expense of persistent serological responses (Schurig et al., 2002).
  • Tradename: RB51
  • Manufacturer: Colorado Serum Company CZ Veterinaria
  • Vaccine Ontology ID: VO_0000021
  • Type: Live attenuated B. abortus strain
  • Status: Licensed
  • Location Licensed: USA, Mexico, South American, India, Spain, Middle East, Iran
  • Host Species for Licensed Use: Cattle
  • Antigen: The antigen for this live attenuated vaccine is the whole cells of strain RB51. This strain has rough characteristics and is devoid of O-chain. The RB51 was derived from the virulent smooth B. abortus 2308 by several passages in media supplemented with sub-inhibitory concentrations of rifampicin (Schurig et al., 1991).
  • Virulence: RB51 is a rough attenuated strain (Schurig et al., 1991).
  • Approved Age for Licensed Use: Calves (4–12 months of age) are vaccinated with RB51 at the full dose of 1.0–3.4 × 1010 colony forming units. In Brucella-infected herds the vaccine can be safely used in cows at a reduced dose of 1.0 × 109 CFU (Poester et al., 2006).
  • Description: RB51 has been the official cattle Brucella vaccine in the USA since 1996 and now in many other countries (Schurig et al., 2002). RB51 is a rifampin-resistant rough attenuated mutant of Brucella abortus derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. Rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop specific antibodies for the Brucella O-chain (Schurig et al., 1991).
Host Response Host Response

Mouse Response

  • Vaccination Protocol: Mice (10/treatment group) were injected i.p. with 0.2 ml of a 0.15 M NaCl saline solution (controls) or with 0.2 ml of saline containing approximately 1x105 cfu of strain 2308 or 1x107 cfu of strain 19 or RB51. Blood samples and spleens were taken from age-matched noninfected control mice and from infected mice at 2, 4, 6, 10, or 20 weeks respectively after infection (Stevens et al., 1994b).
  • Challenge Protocol: No challenge performed.

Mouse Response

  • Host Strain: BALB/c mice
  • Vaccination Protocol: ~4 x 108 CFU
  • Persistence: Inoculation (ip) of RB51 into mice results in a splenic colonization which is cleared within four weeks post infection. RB51 does not revert to smooth colony morphology upon passage in vivo (mice) or in vitro (Schurig et al., 1991).
  • Side Effects: No obvious symptoms were observed in immunized mice.
  • Efficacy: The CFU in spleen decreases 1-2 logs.
  • Description: Vaccination of mice with RB51 induces specific cytotoxic T lymphocytes (CTLs) against strain RB51 and strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. Antigen-specific cytotoxic activity is exerted by T lymphocytes but not by NK cells. CD3+ CD4+ T cells secrete the highest level of IFN-γ and induce low but significant level of lysis of Brucella-infected macrophages. In contrast, CD3+ CD8+ T cells secrete low levels of IFN-γ but high levels of specific lysis of Brucella-infected macrophages. No nonspecific lysis was observed. Hence CD3+ CD4+ and CD3+ CD8+ T cells play synergistic roles in anti-Brucella activity (He et al., 2001).
  • Host Ifng (Interferon gamma) response
    • Description: IFN-gamma is up-regulated in RB51-immunized mice (He et al., 2001).
    • Detailed Gene Information: Click Here.

Mouse Response

  • Host Strain: CD1
  • Vaccination Protocol: Four groups of 10 female CD1 mice per group (5-6 weeks old) were vaccinated intraperitoneally (IP) with 3-5 x 108 CFU in 100 µl of RB51, RB51leuB, RB51leuB/pNS4, or RB51leuB/pNS4/GFP. Another group of 10 mice were vaccinated with saline to serve as a negative control. Three mice from each group were bled at 5 weeks post-vaccination to harvest serum. The sera were screened for GFP-specific antibodies by immunoblot (Rajasekaran et al., 2008).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 RB51 10 in volume female 5.5 week no Vaccinated intraperitoneally (IP) with 3-5 x 108 CFU in 100 µl of RB51. Three mice were bled at 5 weeks post-vaccination to harvest serum, which was screened for GFP-specific antibodies by immunoblot (Rajasekaran et al., 2008).
    2 RB51leuB 10 in volume female 5.5 week no Vaccinated intraperitoneally (IP) with 3-5 x 108 CFU in 100 µl of RB51leuB. Three mice were bled at 5 weeks post-vaccination to harvest serum, which was screened for GFP-specific antibodies by immunoblot (Rajasekaran et al., 2008).
    3 RB51leuB/pNS4 10 in volume female 5.5 week no Vaccinated intraperitoneally (IP) with 3-5 x 108 CFU in 100 µl of RB51leuB/pNS4. Three mice were bled at 5 weeks post-vaccination to harvest serum, which was screened for GFP-specific antibodies by immunoblot (Rajasekaran et al., 2008).
    4 RB51leuB/pNS4/GFP 10 in volume female 5.5 week no Vaccinated intraperitoneally (IP) with 3-5 x 108 CFU in 100 µl of RB51leuB/pNS4/GFP. Three mice were bled at 5 weeks post-vaccination to harvest serum, which was screened for GFP-specific antibodies by immunoblot (Rajasekaran et al., 2008).
    5 Saline 10 in volume female 5.5 week yes Injected intraperitoneally (IP) with 100 µl of saline to act as negative controls. Three mice were bled at 5 weeks post-vaccination to harvest serum, which was screened for GFP-specific antibodies by immunoblot (Rajasekaran et al., 2008).
  • Persistence: Mice vaccinated with the leuB mutant and the complemented RB51leuB strains were able to clear the virulent challenge strain B. abortus 2308 (S2308) at significant rates compared with clearance in the mice vaccinated with saline. No significant difference was observed in the rates of clearance of the challenge strain in the mice vaccinated with auxotrophic vaccine strains and the standard strain RB51 and also between the RB51leuB strains. In a separate experiment, the leuB auxotroph and the complemented leuB auxotroph were found to be cleared from CD1 mouse spleens by 4 to 5 weeks, at the same rate as the parent vaccine strain RB51 (Rajasekaran et al., 2008).
  • Challenge Protocol: At 6 weeks post-vaccination, all groups of mice were challenged IP with 4 x 104 CFU of B. abortus strain 2308 (S2308). At 2 weeks post-challenge, mice were euthanized by CO2 asphyxiation, and their spleens were recovered, homogenized, serially diluted, and plated on trypticase soy agar (TSA) plates to estimate CFU (Rajasekaran et al., 2008).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 40000 CFU in volume 0.1 ml Intraperitoneal injection (i.p.) day 6 week
  • Efficacy: The leuB/GFP construction was shown to complement a leucine auxotroph of RB51, which protected CD1 mice from virulent B. abortus 2308 (S2308) and elicited GFP antibodies. The leuB auxotroph and the complemented auxotroph of strain RB51 were able to protect the CD1 mice against an S2308 challenge. There was no significant difference in the protection levels (as measured by splenic clearance) afforded by the leuB auxotroph when the protection levels in the mice vaccinated with the complemented leuB auxotroph and with the complemented leuB auxotroph expressing GFP were compared to the protection level in the mice vaccinated with strain RB51. There was, however, a significant difference in protection afforded between the mice vaccinated with any of the RB51 strains and the saline control. Only sera from the group inoculated with RB51leuB/pNS4/GFP possessed GFP-specific antibodies (Rajasekaran et al., 2008).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 90000 CFU Each of the RB51 groups (RB51, RB51leuB, RB51leuB/pNS4, and RB51leuB/pNS4GFP) increased clearance by ~ 1 log (104 CFU vs. 105 CFU for saline control group, so reduction of ~90,000 CFU). However, there was no significant difference (NSD) between groups (Rajasekaran et al., 2008).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: To characterize the optimal aerosol dosage of Brucella abortus strain 2308 (S2308) and B. melitensis (S16M), 103–1010 CFU were nebulized to BALB/c mice. A range of dosages was evaluated to determine optimal dosages for aerosolization. Protection in vaccinated and non-vaccinated mice were compared after experimental B. abortus challenge via IP and aerosol routes. For aerosol delivery, the nebulization apparatus consists of a compressed air tank and a jet nebulizer connected via a rubber hose to a port in a metal cage cover. A flow rate of 2 l/min produces a 5 ml particle size. Compressed air was used to jet nebulize 2 ml of challenge inoculum directly into a plexiglass cage containing 5 mice. Nebulization continued until all inoculum, plus 2 ml saline was delivered over ~15 min.
    Experiment 1: S2308 or S16M were spectrophotometrically adjusted to concentrations between 103 and 1010 CFU and delivered by aerosol to female 10-week-old BALB/c mice. At 2 weeks after nebulization, mice were euthanized with CO2. Spleen, liver, and lung from each mouse were removed and weighed. Tissues were macerated and serially diluted, with standard plate counts used to determine bacterial concentrations.
    Experiment 2: Mice were injected IP with 0.2 ml of saline +/- ~1 x 107 CFU of SRB51. At 12 weeks after vaccination, mice were challenged with S2308 IP (0.2 ml saline + 5 x 104 CFU) or via nebulization (~109 CFU). Two weeks after challenge, mice were euthanized with CO2. Spleens, livers and lungs were weighed and processed for isolation and quantification of Brucella as described in Experiment 1.
    (Olsen et al., 2007)
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 Nebulized S2308 CFU 10 in volume female 10 week no Brucella abortus strain 2308 (S2308) was spectrophotometrically adjusted to concentrations between 103 and 1010 CFU and delivered by aerosol to female 10-week-old BALB/c mice. At 2 weeks after nebulization, mice were euthanized with CO2. Spleen, liver, and lung from each mouse were removed and weighed. Tissues were macerated and serially diluted, with standard plate counts used to determine bacterial concentrations (Olsen et al., 2007).
    2 Nebulized S16M CFU 10 in volume female 10 week no Brucella melitensis strain 16M (S16M) were spectrophotometrically adjusted to concentrations between 103 and 1010 CFU and delivered by aerosol to female 10-week-old BALB/c mice. At 2 weeks after nebulization, mice were euthanized with CO2. Spleen, liver, and lung from each mouse were removed and weighed. Tissues were macerated and serially diluted, with standard plate counts used to determine bacterial concentrations (Olsen et al., 2007).
    3 IP saline vaccine 15 in volume female 10 week yes Control mice were injected IP with 0.2 ml of saline. At 12 weeks after vaccination, mice were challenged with S2308 IP (0.2 ml saline + 5 x 104 CFU) or via nebulization (~109 CFU). Two weeks after challenge, mice were euthanized with CO2. Spleens, livers and lungs were weighed and processed for isolation and quantification of Brucella as described in Experiment 1 (Olsen et al., 2007).
    4 IP SRB51 vaccine 15 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice were injected IP with 0.2 ml of saline containing 107 CFU of SRB51. At 12 weeks after vaccination, mice were challenged with S2308 delivered either IP or via nebulization. Two weeks after challenge, mice were euthanized with CO2/O2. Spleens, livers and lungs were weighed and processed for isolation and quantification of Brucella (Olsen et al., 2007).
  • Persistence: Total CFU per tissue increased beginning at 106–107 CFU dosages, with 109 CFU appearing to be an optimal dosage for S16M or S2308 aerosol delivery.
    (Olsen et al., 2007)
  • Immune Response: (Olsen et al., 2007)
  • Side Effects: (Olsen et al., 2007)
  • Challenge Protocol: At 12 weeks after vaccination with 107 CFU of SRB51 or saline (control), mice were challenged IP with 6.4 x 104 CFU or via aerosol (1.76 x 109 CFU) with S2308.
    (Olsen et al., 2007)
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 64000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 12 week
    2 S2308 1760000000 CFU in volume 4 ml nebeulization (aerosol) day 12 week
  • Efficacy: Mice vaccinated with SRB51 had reduced splenic, liver and lung colonization after IP challenge with S2308 as compared with control mice after IP S2308 challenge. Control and SRB51-vaccinated mice did not differ in splenic, liver, or lung colonization after aerosol S2308 challenge.
    (Olsen et al., 2007)
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 2 CFU Mice vaccinated with SRB51 had reduced splenic (decreased by ~2 CFU/g x tissue weight), liver (decreased by ~3 CFU/g x tissue weight), and lung (decreased by ~5 CFU/g x tissue weight) colonization after IP challenge with S2308 as compared with control mice after IP S2308 challenge. Control and SRB51-vaccinated mice did not differ in splenic, liver, or lung colonization after aerosol S2308 challenge (Olsen et al., 2007).
    2 assay of CFU reduction in liver 3 CFU Mice vaccinated with SRB51 had reduced splenic (decreased by ~2 CFU/g x tissue weight), liver (decreased by ~3 CFU/g x tissue weight), and lung (decreased by ~5 CFU/g x tissue weight) colonization after IP challenge with S2308 as compared with control mice after IP S2308 challenge. Control and SRB51-vaccinated mice did not differ in splenic, liver, or lung colonization after aerosol S2308 challenge (Olsen et al., 2007).
    3 assay of CFU redcution in lung 5 CFU Mice vaccinated with SRB51 had reduced splenic (decreased by ~2 CFU/g x tissue weight), liver (decreased by ~3 CFU/g x tissue weight), and lung (decreased by ~5 CFU/g x tissue weight) colonization after IP challenge with S2308 as compared with control mice after IP S2308 challenge. Control and SRB51-vaccinated mice did not differ in splenic, liver, or lung colonization after aerosol S2308 challenge (Olsen et al., 2007).
  • Description: (Olsen et al., 2007)

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: Investigators evaluated the effect of oral inoculation (PO) of Brucella abortus RB51 in BALB/c female mice against a challenge infection with B. abortus (S)2308. For IP immunization with live bacteria, mice were given 2 x 108 CFU of B. abortus RB51 in 0.2 ml of sterile saline or 2 x 104 CFU of strain S2308. For PO immunization, a gastric lavage needle was used. Ten minutes prior to oral inoculation, mice were administered 0.2 ml of 10% sodium bicarbonate (NaHCO3) to neutralize gastric acidity or 0.2 ml of sterile saline. Mice were inoculated PO with 2 x 1010 CFU of RB51 or S2308. On days 10 and 18 after PO RB51 or S2308, preceded or not by gastric acidity neutralization, mice were killed and their spleens removed. Spleens were homogenized in 1 ml of sterile saline. Aliquots of the resulting suspensions were plated to assess spleen colonization.
    In separate experiments, mice were inoculated PO with RB51. Oral inoculation was preceded by gastric acidity neutralization. To evaluate the excretion of bacteria after oral exposure, fecal samples were collected daily for 3 days after inoculation. At 7, 15, 30, and 42 days after infection, mice were bled and killed and spleens were aseptically removed, weighed, homogenized in PBS, and plated to determine the outcome of infection (Pasquali et al., 2003).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 IP RB51 5 in volume female 13 week no For IP immunization with live bacteria, mice were given 2 x 108 CFU of Brucella abortus RB51 in 0.2 ml of sterile saline (Pasquali et al., 2003).
    2 IP S2308 5 in volume female 13 week no For IP immunization with live bacteria, mice were given 2 x 104 CFU of B. abortus 2308 (Pasquali et al., 2003).
    3 IP saline 5 in volume female 13 week yes For negative control of IP immunization, mice were given 0.2 ml of sterile saline (Pasquali et al., 2003).
    4 PO RB51 + NaHCO3 5 in volume female 13 week no For oral (PO) immunization, a gastric lavage needle was used. Ten minutes prior to oral inoculation, mice were administered 0.2 ml of
    10% sodium bicarbonate (NaHCO3) to neutralize gastric acidity. Mice were orally inoculated with 2 x 1010 CFU of RB51. On days 10 and 18 after PO RB51 or S2308, preceded or not by gastric acidity neutralization, mice were killed and their spleens removed. Spleens were homogenized in 1 ml of sterile saline. Aliquots of the resulting suspensions were plated to assess spleen colonization (Pasquali et al., 2003).
    5 PO RB51 + saline 5 in volume female 13 week no For oral (PO) immunization, a gastric lavage needle was used. Ten minutes prior to oral inoculation, mice were administered 0.2 ml of sterile saline. Mice were orally inoculated with 2 x 1010 CFU of RB51. On days 10 and 18 after PO RB51 or S2308, preceded or not by gastric acidity neutralization, mice were killed and their spleens removed. Spleens were homogenized in 1 ml of sterile saline. Aliquots of the resulting suspensions were plated to assess spleen colonization (Pasquali et al., 2003).
    6 PO S2308 + NaHCO3 5 in volume female 13 week no For oral (PO) immunization, a gastric lavage needle was used. Ten minutes prior to oral inoculation, mice were administered 0.2 ml of
    10% sodium bicarbonate (NaHCO3) to neutralize gastric acidity. Mice were orally inoculated with 2 x 1010 CFU of S2308. On days 10 and 18 after PO RB51 or S2308, preceded or not by gastric acidity neutralization, mice were killed and their spleens removed. Spleens were homogenized in 1 ml of sterile saline. Aliquots of the resulting suspensions were plated to assess spleen colonization (Pasquali et al., 2003).
    7 PO S2308 + saline 5 in volume female 13 week no For oral (PO) immunization, a gastric lavage needle was used. Ten minutes prior to oral inoculation, mice were administered 0.2 ml of sterile saline. Mice were orally inoculated with 2 x 1010 CFU of S2308. On days 10 and 18 after PO RB51 or S2308, preceded or not by gastric acidity neutralization, mice were killed and their spleens removed. Spleens were homogenized in 1 ml of sterile saline. Aliquots of the resulting suspensions were plated to assess spleen colonization (Pasquali et al., 2003).
    8 PO saline 5 in volume female 13 week yes For oral (PO) control, a gastric lavage needle was used to inoculate mice with 0.2 ml of sterile saline. On days 10 and 18 after inoculation, mice were killed and their spleens removed. Spleens were homogenized in 1 ml of sterile saline. Aliquots of the resulting suspensions were plated to assess spleen colonization (Pasquali et al., 2003).
  • Persistence: Gastric acid neutralization prior to the oral inoculation contributed to a more homogeneous and consistent infection with both RB51 and S2308. Clearance and immune response following oral infection with RB51 was successively assessed. PO inoculation gave a mild infection, which was cleared 42 days later and induced a delayed humoral and cell-mediated immune (CMI) response (Pasquali et al., 2003).
  • Immune Response: (Pasquali et al., 2003)
  • Side Effects: (Pasquali et al., 2003)
  • Challenge Protocol: Finally, they immunized mice PO with RB51 and challenged them with S2308 PO or intraperitoneally (IP) 42 days after vaccination. Mice were PO vaccinated with RB51 preceded by gastric acidity neutralization or were IP inoculated with RB51 as mentioned above. Additional mice served as unvaccinated controls. PO and IP vaccinated mice and controls were challenged PO (2 x 1010 CFU) or IP (2 x 104 CFU) with S2308 42 days after vaccination. PO challenge was preceded by gastric acid neutralization. Mice were bled and killed at 18 days after challenge. Spleens were weighed and homogenized to determine CFU as a means for assessing the protective response induced by vaccination. Sera were used to evaluate antibody titer against both S2308 and RB51 (Pasquali et al., 2003).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 2000000000 CFU in volume ml oral gavage (actual dose = 2 x 1010) day 42 day
    2 S2308 20000 CFU in volume ml Intraperitoneal injection (i.p.) day 42 day
  • Efficacy: PO RB51 was able to protect mice infected with S2308 PO but not to mice infected IP. Results indicate that oral inoculation of mice with RB51 is able to give protective immunity against oral infection with virulent strains and that this protection seems to rely on an immune response at the mucosal level (Pasquali et al., 2003).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 2.5 CFU Mice were bled and killed at 18 days after challenge. Spleens were weighed and homogenized to determine CFU as a means for assessing the protective response induced by vaccination. PO RB51 was able to protect mice infected with S2308 PO (decreased CFU by 2.5 log) and to mice infected IP (1.5 log decrease). IP RB51 protection was not significant, though CFU was decreased by ~1 log following vaccination via PO and IP. Results indicate that oral inoculation of mice with RB51 is able to give protective immunity against oral infection with virulent strains and that this protection seems to rely on an immune response at the mucosal level (Pasquali et al., 2003).
    2 assay of CFU reduction in spleen 1.5 CFU Mice were bled and killed at 18 days after challenge. Spleens were weighed and homogenized to determine CFU as a means for assessing the protective response induced by vaccination. PO RB51 was able to protect mice infected with S2308 PO (decreased CFU by 2.5 log) and to mice infected IP (1.5 log decrease). IP RB51 protection was not significant, though CFU was decreased by ~1 log following vaccination via PO and IP. Results indicate that oral inoculation of mice with RB51 is able to give protective immunity against oral infection with virulent strains and that this protection seems to rely on an immune response at the mucosal level (Pasquali et al., 2003).
    3 assay of CFU reduction in spleen 1 CFU Mice were bled and killed at 18 days after challenge. Spleens were weighed and homogenized to determine CFU as a means for assessing the protective response induced by vaccination. PO RB51 was able to protect mice infected with S2308 PO (decreased CFU by 2.5 log) and to mice infected IP (1.5 log decrease). IP RB51 protection was not significant, though CFU was decreased by ~1 log following vaccination via PO and IP. Results indicate that oral inoculation of mice with RB51 is able to give protective immunity against oral infection with virulent strains and that this protection seems to rely on an immune response at the mucosal level (Pasquali et al., 2003).
    4 assay of CFU reduction in spleen 1 CFU Mice were bled and killed at 18 days after challenge. Spleens were weighed and homogenized to determine CFU as a means for assessing the protective response induced by vaccination. PO RB51 was able to protect mice infected with S2308 PO (decreased CFU by 2.5 log) and to mice infected IP (1.5 log decrease). IP RB51 protection was not significant, though CFU was decreased by ~1 log following vaccination via PO and IP. Results indicate that oral inoculation of mice with RB51 is able to give protective immunity against oral infection with virulent strains and that this protection seems to rely on an immune response at the mucosal level (Pasquali et al., 2003).
  • Description: (Pasquali et al., 2003)

Mouse Response

  • Host Strain: C57BL/6 (IRF-1-/-)
  • Vaccination Protocol: S2308 (the virulent wild-type Brucella abortus strain), S19 and RB51 (live attenuated vaccine strains), cyd and cbp (insertional mutants), and bap (deletion mutant) were used in this experiments. Interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. IRF1-/- mice were injected IP with diverse strains and CFU of B. abortus in 200 ul of PBS (Ko et al., 2002).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 IP S19 (5 x 101) 5 in volume female 7.5 week no 6- to 9-week-old interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. Mice were injected IP with Brucella abortus strain S19 in 200 ul of PBS. After 6 weeks of infection, surviving mice were challenged with 5 x 105 CFU of B. abortus S2308 in 200 ul of PBS (Ko et al., 2002).
    2 IP S19 (5 x 103) 5 in volume female 7.5 week no 6- to 9-week-old interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. Mice were injected IP with Brucella abortus strain S19 (5000 CFU) in 200 ul of PBS. After 6 weeks of infection, surviving mice were challenged with 5 x 105 CFU of B. abortus S2308 in 200 ul of PBS (Ko et al., 2002).
    3 IP RB51 (5 x 105) 5 in volume female 7.5 week no 6- to 9-week-old interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. Mice were injected IP with Brucella abortus strain RB51 (5 x 105) in 200 ul of PBS. After 6 weeks of infection, surviving mice were challenged with 5 x 105 CFU of B. abortus S2308 in 200 ul of PBS (Ko et al., 2002).
    4 IP RB51 (5 x 107 CFU) 5 in volume female 7.5 week no 6- to 9-week-old interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. Mice were injected IP with Brucella abortus strain RB51 (5 x 107 CFU) in 200 ul of PBS. After 6 weeks of infection, surviving mice were challenged with 5 x 105 CFU of B. abortus S2308 in 200 ul of PBS (Ko et al., 2002).
    5 IP cyd mutant (5 x 105 CFU) 5 in volume female 7.5 week no 6- to 9-week-old interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. Mice were injected IP with Brucella abortus cyd mutant (5 x 105 CFU) in 200 ul of PBS. After 6 weeks of infection, surviving mice were challenged with 5 x 105 CFU of B. abortus S2308 in 200 ul of PBS (Ko et al., 2002).
    6 IP cyd mutant (5 x 107 CFU) 5 in volume female 7.5 week no 6- to 9-week-old interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. Mice were injected IP with Brucella abortus cyd mutant (5 x 107 CFU) in 200 ul of PBS. After 6 weeks of infection, surviving mice were challenged with 5 x 105 CFU of B. abortus S2308 in 200 ul of PBS (Ko et al., 2002).
    7 IP saline 5 in volume female 7.5 week yes 6- to 9-week-old interferon regulatory factor 1-deficient (IRF1-/-) mice were originally produced by using C57BL/6 (H-2b) mice. Mice were injected IP with 200 ul of PBS. After 6 weeks of infection, surviving mice were challenged with 5 x 105 CFU of B. abortus S2308 in 200 ul of PBS (Ko et al., 2002).
  • Persistence: To count residual CFU in the spleens or livers of mice, 5 mice from each group were examined at each sampling period. Brucella colonies were counted after a 3-day incubation. Serum glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were also quantitatively measured (Ko et al., 2002).
  • Immune Response: (Ko et al., 2002)
  • Challenge Protocol: After 6 weeks, surviving IRF1-/- mice were challenged with 5 x 105 CFU of S2308 in 200 ul of PBS (Ko et al., 2002).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 500000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 6 week
  • Efficacy: Interferon regulatory factor 1-deficient (IRF1-/-) mice infected with virulent Brucella abortus (S)2308 at 5 x 105 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF1-/- mice were unable to resolve infection and died. In contrast, IRF1-/- mice survived when infected at 5 x 105 CFU with several attenuated Brucella strains (S19, RB51, cbp, and cyd). Brucella-infected mice were also killed 12 days post-infection for histologic analysis (Ko et al., 2002).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 % survival of S19 at 50 CFU 80 CFU Interferon regulatory factor 1-deficient (IRF1-/-) mice infected with virulent Brucella abortus (S)2308 at 5 x 105 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF1-/- mice were unable to resolve infection and died. In contrast, IRF1-/- mice survived when infected at 5 x 105 CFU with attenuated Brucella strains S19 50 CFU (80% survival) and 5000 CFU (87.5%), RB51 at 500000 (62.5-87.5%) and 50000000 CFU (100%), and cyd mutant at 500000 (40-80%) and 50000000 CFU (90%). Brucella-infected mice were also killed 12 days post-infection for histologic analysis (Ko et al., 2002).
    2 % survival of S19 at 5000 CFU 87.5 CFU Interferon regulatory factor 1-deficient (IRF1-/-) mice infected with virulent Brucella abortus (S)2308 at 5 x 105 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF1-/- mice were unable to resolve infection and died. In contrast, IRF1-/- mice survived when infected at 5 x 105 CFU with attenuated Brucella strains S19 50 CFU (80% survival) and 5000 CFU (87.5%), RB51 at 500000 (62.5-87.5%) and 50000000 CFU (100%), and cyd mutant at 500000 (40-80%) and 50000000 CFU (90%). Brucella-infected mice were also killed 12 days post-infection for histologic analysis (Ko et al., 2002).
    3 % survival of RB51 at 500000 CFU 62.5-87.5 CFU Interferon regulatory factor 1-deficient (IRF1-/-) mice infected with virulent Brucella abortus (S)2308 at 5 x 105 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF1-/- mice were unable to resolve infection and died. In contrast, IRF1-/- mice survived when infected at 5 x 105 CFU with attenuated Brucella strains S19 50 CFU (80% survival) and 5000 CFU (87.5%), RB51 at 500000 (62.5-87.5%) and 50000000 CFU (100%), and cyd mutant at 500000 (40-80%) and 50000000 CFU (90%). Brucella-infected mice were also killed 12 days post-infection for histologic analysis (Ko et al., 2002).
    4 % survival of RB51 at 50000000 CFU 100 CFU Interferon regulatory factor 1-deficient (IRF1-/-) mice infected with virulent Brucella abortus (S)2308 at 5 x 105 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF1-/- mice were unable to resolve infection and died. In contrast, IRF1-/- mice survived when infected at 5 x 105 CFU with attenuated Brucella strains S19 50 CFU (80% survival) and 5000 CFU (87.5%), RB51 at 500000 (62.5-87.5%) and 50000000 CFU (100%), and cyd mutant at 500000 (40-80%) and 50000000 CFU (90%). Brucella-infected mice were also killed 12 days post-infection for histologic analysis (Ko et al., 2002).
    5 % survival of cyd at 50000000 CFU 40-80 CFU Interferon regulatory factor 1-deficient (IRF1-/-) mice infected with virulent Brucella abortus (S)2308 at 5 x 105 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF1-/- mice were unable to resolve infection and died. In contrast, IRF1-/- mice survived when infected at 5 x 105 CFU with attenuated Brucella strains S19 50 CFU (80% survival) and 5000 CFU (87.5%), RB51 at 500000 (62.5-87.5%) and 50000000 CFU (100%), and cyd mutant at 500000 (40-80%) and 50000000 CFU (90%). Brucella-infected mice were also killed 12 days post-infection for histologic analysis (Ko et al., 2002).
    6 % survival of cyd at 50000000 CFU 90 CFU Interferon regulatory factor 1-deficient (IRF1-/-) mice infected with virulent Brucella abortus (S)2308 at 5 x 105 CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF1-/- mice were unable to resolve infection and died. In contrast, IRF1-/- mice survived when infected at 5 x 105 CFU with attenuated Brucella strains S19 50 CFU (80% survival) and 5000 CFU (87.5%), RB51 at 500000 (62.5-87.5%) and 50000000 CFU (100%), and cyd mutant at 500000 (40-80%) and 50000000 CFU (90%). Brucella-infected mice were also killed 12 days post-infection for histologic analysis (Ko et al., 2002).
  • Description: (Ko et al., 2002)

Mouse Response

  • Host Strain: BALB/c, DBA/2
  • Vaccination Protocol: Young (2–3 month old) or old (16–18 month old) adult female mice (DBA/2 or BALB/c) were infected with either an attenuated Brucella abortus strain that over-expressed superoxide dismutase (RB51-SOD) or with a fully virulent wild-type strain (S2308). They were inoculated intra-peritoneally (IP) with 3.4 x 107 CFU or 6 x 108 CFU RB51-SOD, or with 2 x 104 or 4 x 109 CFU S2308. Uninfected control mice were injected with saline alone. Spleens harvested after sacrifice were cultured quantitatively for Brucella. Splenocytes (80–120 x 106) from mice were harvested and cultured in the presence of heat-killed B. abortus, purified SOD, or the mitogen concanavalin A (conA). Supernatants were assayed for the presence of cytokines by protein array and ELISA, semi-quantitatively measured by densitometry and the intensity of a given dot-blot compared to the mean of the positive control dots. Blood was obtained via retro-orbital bleeds. Total IgG, IgG1 and IgG2a titers vs. two Brucella proteins, bacterioferritin (BFR) and Cu++/Zn++ SOD, were assessed by ELISA (High et al., 2007).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 Young RB51-SOD (3.4 x 107 CFU) 5 in volume female 2 month no Young adult female mice were infected with attenuated Brucella abortus that over-expresses superoxide dismutase (RB51-SOD). They were inoculated intra-peritoneally (IP) with 3.4 x 107 CFU. Spleens harvested after sacrifice were cultured quantitatively for brucellae (High et al., 2007).
    2 Old RB51-SOD (3.4 x 107 CFU) 5 in volume female 18 month no Old (18 month old) adult female mice (DBA/2 or BALB/c) were infected with attenuated Brucella abortus that over-expressed superoxide dismutase (RB51-SOD). They were inoculated intra-peritoneally (IP) with 3.4 x 107 CFU (High et al., 2007).
    3 Young RB51-SOD (6 x 108 CFU) 5 in volume female 2 month no Young (2 month old) adult female mice (DBA/2 or BALB/c) were infected with an attenuated Brucella abortus strain that over-expressed superoxide dismutase (RB51-SOD). They were inoculated intra-peritoneally (IP) with 6 x 108 CFU (High et al., 2007).
    4 Old RB51-SOD (6 x 108 CFU) 5 in volume female 18 month no Old (18 month old) adult female mice (DBA/2 or BALB/c) were infected with an attenuated Brucella abortus strain that over-expressed superoxide dismutase (RB51-SOD). They were inoculated intra-peritoneally (IP) with 6 x 108 CFU RB51-SOD (High et al., 2007).
    5 Young S2308 (4 x 104 CFU) 5 in volume female 2 month no Young (2 month old) adult female mice (DBA/2 or BALB/c) were infected with a fully virulent wild-type Brucella abortus strain (S2308). They were inoculated intra-peritoneally (IP) with 4 x 104 CFU S2308 (High et al., 2007).
    6 Old S2308 (4 x 104 CFU) 5 in volume female 18 month no Old (18 month old) adult female mice (DBA/2 or BALB/c) were infected with a fully virulent wild-type Brucella abortus strain (S2308). They were inoculated intra-peritoneally (IP) with 4 x 104 CFU S2308 (High et al., 2007).
    7 Young S2308 (2 x 104 CFU) 9 in volume female 2 month no Young (2 month old) adult female mice (DBA/2 or BALB/c) were infected with a fully virulent wild-type Brucella abortus strain (S2308). They were inoculated intra-peritoneally (IP) with 2 x 104 CFU S2308 (High et al., 2007).
    8 Old S2308 (2 x 104 CFU) 9 in volume female 17 month no Old (16–18 month old) adult female mice (DBA/2 or BALB/c) were infected with a fully virulent wild-type Brucella abortus strain (S2308). They were inoculated intra-peritoneally (IP) with 2 x 104 CFU S2308 (High et al., 2007).
    9 Young S2308 (2 x 106 CFU) 9 in volume female 2 month no Young (2 month old) adult female mice (DBA/2 or BALB/c) were infected with a fully virulent wild-type Brucella abortus strain (S2308). They were inoculated intra-peritoneally (IP) with 2 x 106 (High et al., 2007).
    10 Old S2308 (2 x 106 CFU) 9 in volume female 17 month no Old (16–18 month old) adult female mice (DBA/2 or BALB/c) were infected with a fully virulent wild-type Brucella abortus strain (S2308). They were inoculated intra-peritoneally (IP) with 2 x 106 CFU S2308 (High et al., 2007).
    11 Young saline 5 in volume female 2 month yes Young (2–3 month old) adult female control mice (DBA/2 or BALB/c) were inoculated intra-peritoneally (IP) with saline alone (High et al., 2007).
    12 Old saline 5 in volume female 18 month yes Old (16–18 month old) adult female control mice (DBA/2 or BALB/c) were inoculated intra-peritoneally (IP) with saline alone (High et al., 2007).
  • Persistence: All young and old mice survived infection with RB51-SOD (up to 6 x 1^08 CFU) or S2308 (up to 8 x 108 CFU). Old mice had a lower organism burden in the spleen than young mice 5 or more weeks after infection (High et al., 2007).
  • Immune Response: Antibody and cytokine responses were Th1-focused in young mice, but Th-mixed in old mice, including evidence of the Th17 subtype immune response (High et al., 2007).
  • Challenge Protocol: For protection studies, mice were either immunized with 4 x 108 CFU RB51-SOD or saline as a control and allowed to recover for 5 weeks. Mice were then infected with 2 x 104 CFU S2308 and sacrificed 2 weeks later for determination of CFU/spleen as above (High et al., 2007).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 20000 CFU in volume ml Intraperitoneal injection (i.p.) day 5 week
  • Efficacy: Immunization with the RB51-SOD strain provided protection vs. strain 2308 challenge in young and aged BALB/c, but only young DBA/2 mice (High et al., 2007).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 1.5 CFU Mean ± SEM cfu/spleen in old and young adult DBA/2 or BALB/c mice two weeks after infection with S2308 in mice that were either naıve or immunized with RB51-SOD five weeks prior to infection with strain 2308. Immunization with the RB51-SOD strain provided protection vs. strain 2308 challenge in young (~1.5 log decrease) and aged BALB/c (~2 log), but only young (~1 log) not in aged (<1 log) DBA/2 mice .Antibody and cytokine responses were Th1-focused in young mice, but Th-mixed in old mice, including evidence of the Th17 subtype immune response (High et al., 2007).
    2 assay of CFU reduction in spleen 2 CFU Mean ± SEM cfu/spleen in old and young adult DBA/2 or BALB/c mice two weeks after infection with S2308 in mice that were either naıve or immunized with RB51-SOD five weeks prior to infection with strain 2308. Immunization with the RB51-SOD strain provided protection vs. strain 2308 challenge in aged BALB/c (~2 log), but only young (~1 log) not in aged (<1 log) DBA/2 mice .Antibody and cytokine responses were Th1-focused in young mice, but Th-mixed in old mice, including evidence of the Th17 subtype immune response (High et al., 2007).
    3 assay of CFU reduction in spleen 1 CFU Mean ± SEM cfu/spleen in old and young adult DBA/2 or BALB/c mice two weeks after infection with S2308 in mice that were either naıve or immunized with RB51-SOD five weeks prior to infection with strain 2308. Immunization with the RB51-SOD strain provided protection vs. strain 2308 challenge only in young (~1 log) not in aged (<1 log) DBA/2 mice (High et al., 2007).
    4 assay of CFU reduction in spleen 0.3 CFU Mean ± SEM cfu/spleen in old and young adult DBA/2 or BALB/c mice two weeks after infection with S2308 in mice that were either naıve or immunized with RB51-SOD five weeks prior to infection with strain 2308. Immunization with the RB51-SOD strain did not provide protection vs. strain 2308 challenge in aged (<1 log) DBA/2 mice .Antibody and cytokine responses were Th1-focused in young mice, but Th-mixed in old mice, including evidence of the Th17 subtype immune response (High et al., 2007).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 RB51 5 in volume female 10 week no Female BALB/c mice (10 weeks old) mice were divided into three groups; the first group was injected intraperitonealy (IP) with 5 x 108 CFU of the live rough Brucella abortus RB51 vaccine (Hamdy et al., 2002).
    2 Rev1 5 in volume female 10 week no Female BALB/c mice (10 weeks old) mice were divided into three groups; the second group was inoculated intraperitonealy (IP) with 5 x 108 CFU with smooth B. melitensis Rev. 1 vaccine (Hamdy et al., 2002).
    3 PBS 5 in volume female 10 week yes Female BALB/c mice (10 weeks old) mice were divided into three groups; the third group was inoculated intraperitonealy (IP) with 0.2 ml of sterile PBS and kept as a control (Hamdy et al., 2002).
  • Challenge Protocol: Mice were challenged with a B. melitensis field strain. The humoral immune responses induced in mice after vaccination and challenge were detected by the standard tube agglutination test (SAT), mercaptoethanol test (MET), Rose Bengal plate test (RBPT), and buffered acidified plate antigen test (BAPAT). The detection of immune responses induced by the RB51 vaccine was measured by RB51 killed antigen. Mice were bled under anesthesia from the retroorbital sinus or from heart puncture. Sera were stored at -70C unless used on the same day. Five animals from each of the first and the second groups were sacrificed by CO2 asphyxiation at weekly intervals. Detection of Brucella vaccinal strains in internal organs (spleen, liver and lung) was done at weekly intervals. Two weeks after elimination of the vaccinal strains from the internal organs and disappearance of any serological reactions, the remaining animals and control were challenged with 5 x 105 cfu of B. melitensis i.p. in 0.2 ml PBS. Five of the challenged animals from each group were sacrificed every week until the end of the experiment. Sacrificed animals were subjected to serological and bacteriological investigations. Bacteriological examinations and viable counts of the Brucella organisms per spleen following challenge were carried out. Identification of the rough form of the RB51 vaccinal strain was achieved by auto-agglutination reaction with acriflavine solution (1/1000) and the ability to stain with crystal violet (Hamdy et al., 2002).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 B. melitensis field strain bv. 3 500000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 2 week
  • Efficacy: Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice vaccinated with RB51 gave negative results in the conventional serological tests prior to challenge, standard tube agglutination test (SAT), Rose Bengal plate test (RBPT), buffered acidified plate antigen test (BAPAT), and mercaptoethanol test (MET). Sero-conversion took place to a whole-cell bacterial buffered RB51 antigen after vaccination and persisted for 7 weeks post-vaccination. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Protection units were showed that Rev. 1 vaccine was superior to RB51 vaccine in protection of mice against B. melitensis (Hamdy et al., 2002).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 1.14 CFU Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Log units of protection (decrease in splenic CFU) were measured at 2 (1.14 log decrease), 3 (1.5 log), 4 (~2 log), 5 (<1 log), 10 (<1 log), and 12 weeks (~1 log) (Hamdy et al., 2002).
    2 assay of CFU reduction in spleen 1.5 CFU Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Log units of protection (decrease in splenic CFU) were measured at 3 (1.5 log), 4 (~2 log), 5 (<1 log), 10 (<1 log), and 12 weeks (~1 log) (Hamdy et al., 2002).
    3 assay of CFU reduction in spleen 2 CFU Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Log units of protection (decrease in splenic CFU) were measured at 4 (~2 log), 5 (<1 log), 10 (<1 log), and 12 weeks (~1 log) (Hamdy et al., 2002).
    4 assay of CFU reduction in spleen 0.7 CFU Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Log units of protection (decrease in splenic CFU) were measured at 5 (<1 log), 10 (<1 log), and 12 weeks (~1 log) (Hamdy et al., 2002).
    5 assay of CFU reduction in spleen 0.5 CFU Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Log units of protection (decrease in splenic CFU) were measured at 10 (<1 log) and 12 weeks (~1 log) (Hamdy et al., 2002).
    6 assay of CFU reduction in spleen 1 CFU Protection was assessed by a variety of serological tests and recovery of vaccinal and challenge strains by culture. Mice challenged with B. melitensis were assessed for bacterial load and immune response for 12 weeks after challenge. Log units of protection (decrease in splenic CFU) were measured at 12 weeks (~1 log) (Hamdy et al., 2002).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: This study determined if murine interleukin-12 (IL-12) would influence immunity in mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). Mice received live or irradiated SRB51 bacteria alone, or with IL-12 (0.5 or 1.0 jig, 2x or 3X), whereas other mice received saline or IL-12 alone. Specifically, female 10-week-old BALB/c AnNHsD mice (n = 12/group) were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 3 x 108 CFU of live SRB51 alone, or combined with 0.5 or 1.0 ug of murine recombinant IL-12, administered thrice (days 0, 5, and 21). Additional mice were vaccinated IP with 0.2 mL of saline containing 3 x 108 CFU of irradiated SRB51 alone, or combined with 0.5 or 1.0 ug of IL-12 administered twice (days 0 and 5) or thrice. Other mice were injected IP with 0.2 mL of saline alone or with saline containing 0.5 or 1.0 ug of IL-12 administered twice or thrice (Lee et al., 2001).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 live SRB51 12 in volume female 10 week no Mice received live or irradiated Brucella abortus strain RB51 (SRB51) bacteria alone, or with murine interleukin-12 (IL-12) (0.5 or 1.0 ug, 2x or 3x), whereas other mice received saline or IL-12 alone. Specifically, female 10-week-old BALB/c AnNHsD mice (n = 12/group) were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 3 x 108 CFU of live SRB51 alone (Lee et al., 2001).
    2 live SRB51 + 0.5 ug IL-12 12 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice (n = 12/group) were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 3 x 108 CFU of live SRB51 combined with 0.5 ug of murine recombinant IL-12 administered thrice (Lee et al., 2001).
    3 live SRB51 + 1.0 ug IL-12 12 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice (n = 12/group) were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 3 x 108 CFU of live SRB51 combined with 1.0 ug of murine recombinant IL-12, administered thrice (days 0, 5, and 21). Additional mice were vaccinated IP with 0.2 mL of saline containing 3 x 108 CFU of irradiated SRB51 alone, or combined with 0.5 or 1.0 ug of IL-12 administered twice (days 0 and 5) or thrice. Other mice were injected IP with 0.2 mL of saline alone or with saline containing 0.5 or 1.0 ug of IL-12 administered twice or thrice (Lee et al., 2001).
    4 killed SRB51 8 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 3 x 108 CFU irradiated SRB51 alone (Lee et al., 2001).
    5 killed SRB51 + 0.5 ug IL-12 8 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 3 x 108 CFU of irradiated SRB51 combined with 0.5 ug of IL-12 administered twice (days 0 and 5) or thrice (Lee et al., 2001).
    6 killed SRB51 + 1.0 ug IL-12 12 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 3 x 108 CFU of irradiated SRB51 combined with 1.0 ug of IL-12 administered twice (days 0 and 5) or thrice (Lee et al., 2001).
    7 saline 12 in volume female 10 week yes Female 10-week-old BALB/c AnNHsD mice were inoculated intraperitoneally (IP) with 0.2 mL of saline alone (Lee et al., 2001).
    8 saline + 0.5 ug IL-12 8 in volume female 10 week yes Female 10-week-old BALB/c AnNHsD mice were inoculated intraperitoneally (IP) with 0.2 mL of saline containing 0.5 ug of IL-12 administered twice or thrice (Lee et al., 2001).
    9 saline + 1.0 ug IL-12 8 in volume female 10 week yes Female 10-week-old BALB/c AnNHsD mice were inoculated intraperitoneally (IP) with 0.2 mL of saline containing 1.0 ug of IL-12 administered twice or thrice (Lee et al., 2001).
  • Persistence: Post-vaccination antibody responses to live or killed SRB51 and clearance of live SRB51 from splenic tissue were not influenced by IL-12 treatments (Lee et al., 2001).
  • Challenge Protocol: Mice were challenged at 12 weeks with 4 x 104 CFU of B. abortus strain 2308 (S2308) and were euthanized 2 weeks later. Remaining mice (n = 8/trt) were challenged IP with 4 X 104 cfu of S2308 at 12 wk after vaccination. The time of challenge was based on data from previous studies which demonstrated that a dosage of 5 X 108 cfu of SRB51 is cleared from BALB/c mice by 12 wk after IP vaccination (Lee et al., 2001).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 40000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 12 week
  • Efficacy: The highest IL-12 treatment increased post-challenge antibody responses when co-administered with killed SRB51. Co-administration of 1.0 ug of IL-12 with live SRB51, but not killed SRB51, reduced S2308 colonization of splenic tissues. Following euthanasia with C02/02 at 2 wk after S2308 challenge, blood, spleens, and livers were obtained from all mice. Spleens were weighed, approximately onehalf was excised for bacteriologic examination and weighed, and remaining tissue was used to prepare spleen cell suspensions. Following weighing, the entire liver was designated for bacteriologic examination. Tissue for bacteriologic examination was stored at -69°C until processed (Lee et al., 2001).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 0.5-1.5 CFU Co-administration of IL-12 with live SRB51, but not killed SRB51, reduced S2308 colonization of splenic tissues. Colonization of splenic and hepatic tissues as cfu (log1o) at 2 weeks after IP challenge with 4 X 104 cfu of S2308. Mice had been vaccinated 12 wk prior to challenge with live or killed SRB51, alone, or in combination with IL-12, or, with saline or IL-12 alone. CFU/g spleen was decreased less in mice vaccinated with live (~1 log less) or killed (0.5 log) SRB51 alone than in mice given IL-12 (~1.5 log) (Lee et al., 1999).
    2 assay of CFU reduction in liver 1.5 CFU Co-administration of IL-12 with live SRB51, but not killed SRB51, reduced S2308 colonization of hepatic tissues. Colonization of tissues as cfu (log1o) at 2 weeks after IP challenge with 4 X 104 cfu of S2308. Mice had been vaccinated 12 wk prior to challenge with live or killed SRB51, alone, or in combination with IL-12, or, with saline or IL-12 alone. CFU/g liver was decreased significantly in mice vaccinated with live SRB51 +/- IL-12 (~1.5 log less) but not in mice given killed SRB51 (Lee et al., 1999).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: The present study was performed to delineate cytokine induction after intraperitoneal (IP) administration of Brucella abortus strain RB51 and subsequent infection with virulent strain B. abortus S2308. Twelve- to 14-week-old BALB/c female mice were allocated to experimental groups consisting of five animals each. Mice were vaccinated IP with 0.2 ml of saline containing 2 x 108 CFU of RB51. Unvaccinated control animals remained untreated throughout the experiment (Pasquali et al., 2001).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 RB51 5 in volume female 13 week no Twelve- to 14-week-old BALB/c female mice were vaccinated IP with 0.2 ml of saline containing 2 x 108 CFU of RB51 (Pasquali et al., 2001).
    2 Saline control 5 in volume female 13 week yes Twelve- to 14-week-old unvaccinated control BALB/c female mice were inoculated IP with 0.2 ml of saline and remained untreated throughout the experiment (Pasquali et al., 2001).
  • Persistence: Vaccinated mice had higher spleen weights when compared to control animals after vaccination. Vaccination of mice with live RB51 resulted in a pattern of bacterial growth in which peak numbers were seen 18 days after vaccination, followed by a progressive decline. Bacteria were absent in the spleen at 42 days postvaccination. Counts on days 6 and 18 were significantly different (Pasquali et al., 2001).
  • Immune Response: 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. Spleen weight in vaccinated and infected mice did not increase after challenge and were not significantly different from nonchallenged, vaccinated animals killed 42 days after vaccination. In contrast, spleen weights of unvaccinated and infected mice showed a significant enlargement as early as 6 days after infection. Vaccinated mice were almost refractory to a subsequent challenge infection with B. abortus 2308 cells, exhibiting significantly lower levels of infection compared to unvaccinated infected animals after infection. Vaccination alone induced a number of significant effects upon cytokine expression. Spleen cells from unvaccinated mice stimulated with heat-inactivated RB51 cells did not produce measurable cytokines throughout the course of the experiment. Similarly, spleen cells from vaccinated mice killed 6 and 18 days after vaccination did not produce measurable bio-active IL-12 (p70) when stimulated in vitro, but a low level of production was detectable in spleen cells from mice killed 42 days after vaccination (Pasquali et al., 2001).
  • Challenge Protocol: At 42 days after vaccination, unvaccinated controls and IP vaccinated mice were challenged IP with 0.2 ml of saline containing 2 x 104 CFU of B. abortus 2308. At 6, 18, and 42 days after vaccination and 3, 6, and 10 days after challenge, mice were euthanatized and spleens were aseptically removed. Approximately one-third of the spleen was weighed and homogenized in PBS, and an aliquot of the resulting cell suspension was plated to determine the number of CFU. The remaining 2/3 of the spleens were weighed, minced, and used to prepare spleen cell suspensions. Cytokine expression was assayed in culture supernatants of splenocytes stimulated with 108 heat-inactivated B. abortus RB51 or 2308 bacteria per well. Cytokines were detected by ELISA according to the manufacturer’s instructions (Pasquali et al., 2001).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 20000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 42 day
  • Efficacy: RB51 confers a solid immunity that protects mice against a challenge infection with virulent B. abortus 2308. After vaccination, both Th1 and Th2 cytokine patterns were observed. Protection may be more dependent upon the timing of the cytokine response rather than on the absolute level of cytokine expression. Challenge infection of vaccinated and nonvaccinated mice induced even more marked increases in cytokine expression patterns. IL-12 p40 was detectable in spleen cells from both vaccinated and unvaccinated mice after challenge infection, and there were no significant differences between vaccinated and unvaccinated mice. Similar results were seen throughout the course of the experiment for IL-12 p70 production, which was low at all times in all groups of mice. In contrast, IFN- production was detectable as early as 3 days after challenge infection in spleen cells from both vaccinated and unvaccinated mice, although levels were significantly higher in vaccinated mice. Levels of IFN in spleen cells from vaccinated, challenged mice showed no differences throughout the experiment, in contrast to spleen cells from unvaccinated, challenged mice which showed increased IFN production that reached levels seen in spleen cells from vaccinated, challenged mice 6 days after challenge. After challenge infection, low levels of IL-4 were detectable, reaching a peak in spleen cells 6 days after challenge. However, levels were not different between vaccinated and unvaccinated mice. IL-10 showed a pattern similar to that of IFN-. IL-10 was detectable in both vaccinated and unvaccinated mice after challenge infection. However, vaccinated and challenged animals did not show any differences at different times after challenge. Conversely, IL-10 production in unvaccinated, challenged mice was first detectable 6 days after challenge infection and reached a peak 10 days after challenge infection. Results of this study indicated that mice vaccinated with RB51 cells were protected as early as 3 days after a challenge infection with virulent B. abortus 2308 cells. In vaccinated mice, challenge infection resulted in high levels of IL-10 production at all times tested. In contrast, in unvaccinated mice, IL-10 production reached levels comparable to that in the vaccinated mice beginning 6 days after infection. Previous studies indicate that both IL-10 and IFN- are produced following a B. abortus infection and that IL-10 induction does not down-regulate IFN- production. This implies that in brucellosis, the effect of IL-10 on the immune response is to limit the consequences of an exaggerated proinflammatory response more than to counterbalance the production of Th1 cytokines. These findings are in agreement with others which measured IL-10 production following Brucella infection (Pasquali et al., 2001).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 1.1 CFU At 3, 6, and 10 days after challenge, mice were euthanatized and spleens were aseptically removed and used to determine the number of CFU and cytokine expressionvia ELISA. RB51 confers a solid immunity that protects mice against a challenge infection with virulent B. abortus 2308. Both Th1 and Th2 cytokine patterns were observed. Challenge infection of vaccinated and nonvaccinated mice induced even more marked increases in cytokine expression patterns. Persistence of B. abortus S2308 in spleens of vaccinated and unvaccinated mice was also analyzed as splenic CFU decrease at 3 days (1.1 log less than unvaccinated controls), 6 days (2.2 log less), and 10 days (2.4 log) post-challenge (Pasquali et al., 2001).
    2 assay of CFU reduction in spleen 2.2 CFU At 3, 6, and 10 days after challenge, mice were euthanatized and spleens were aseptically removed and used to determine the number of CFU and cytokine expressionvia ELISA. RB51 confers a solid immunity that protects mice against a challenge infection with virulent B. abortus 2308. Both Th1 and Th2 cytokine patterns were observed. Challenge infection of vaccinated and nonvaccinated mice induced even more marked increases in cytokine expression patterns. Persistence of B. abortus S2308 in spleens of vaccinated and unvaccinated mice was also analyzed as splenic CFU decrease at 3 days (1.1 log less than unvaccinated controls), 6 days (2.2 log less), and 10 days (2.4 log) post-challenge (Pasquali et al., 2001).
    3 assay of CFU reduction in spleen 2.4 CFU At 3, 6, and 10 days after challenge, mice were euthanatized and spleens were aseptically removed and used to determine the number of CFU and cytokine expressionvia ELISA. RB51 confers a solid immunity that protects mice against a challenge infection with virulent B. abortus 2308. Both Th1 and Th2 cytokine patterns were observed. Challenge infection of vaccinated and nonvaccinated mice induced even more marked increases in cytokine expression patterns. Persistence of B. abortus S2308 in spleens of vaccinated and unvaccinated mice was also analyzed as splenic CFU decrease at 3 days (1.1 log less than unvaccinated controls), 6 days (2.2 log less), and 10 days (2.4 log) post-challenge (Pasquali et al., 2001).

Mouse Response

  • Host Strain: Deer mouse (Peromyscus maniculatus)
  • Vaccination Protocol: This study was designed to determine effects of strain RB51 on deer mice (Peromyscus maniculatus), a nontarget species that could have access to treated baits in a field situation. 90 mice were orally dosed (PO) or intraperitoneally injected (IP) with 1 x 108 colony forming units (CFU) strain RB51, and 77 controls were similarly dosed with sterile saline. At weekly intervals for 2 months, 4-6 mice from each group were euthanized, gross necropsies performed, spleens and uteruses cultured, and tissues examined histologically (Cook et al., 2001).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 IP saline 37 in volume female 3-5 month yes One hundred eighty captive raised 3–5 mo-old deer mice were randomly divided into four groups of 45 each containing approximately equal numbers of males and nonpregnant females. Mice were given a 2 wk acclimation period prior to beginning the experiment. To allow comparison of oral inoculation (PO) with intraperitoneal inoculation (IP), the standard test method in laboratory mice, both IP and PO experimental groups were established. Because some mortality occurred during the acclimation period, some groups had fewer than 45 mice remaining. One group (n = 37) of mice was injected IP with 0.2 ml of 0.85% sterile saline pH 7.0. Every week for 8 wk beginning 1 wk post inoculation (WPI) 4-6 mice were euthanized via cervical dislocation and necropsied (Cook et al., 2001).
    2 PO saline 40 in volume female 3-5 month yes One hundred eighty captive raised 3–5 mo-old deer mice were randomly divided into four groups of 45 each containing approximately equal numbers of males and nonpregnant females. Mice were given a 2 wk acclimation period prior to beginning the experiment. To allow comparison of oral inoculation (PO) with intraperitoneal inoculation (IP), the standard test method in laboratory mice, both IP and PO experimental groups were established. Because some mortality occurred during the acclimation period, some groups had fewer than 45 mice remaining. One group of mice (n = 40) was orally dosed with 0.2 ml of 0.85% saline via a tuberculin syringe (without a needle). Every week for 8 wk beginning 1 wk post inoculation (WPI) 4-6 mice were euthanized via cervical dislocation and necropsied (Cook et al., 2001).
    3 IP RB51 45 in volume female 3-5 month no One hundred eighty captive raised 3–5 mo-old deer mice were randomly divided into four groups of 45 each containing approximately equal numbers of males and nonpregnant females. Mice were given a 2 wk acclimation period prior to beginning the experiment. To allow comparison of oral inoculation (PO) with intraperitoneal inoculation (IP), the standard test method in laboratory mice, both IP and PO experimental groups were established. Because some mortality occurred during the acclimation period, some groups had fewer than 45 mice remaining. One group of mice (n = 45) was injected IP with 0.2 ml of 85% sterile saline pH 7.0 containing 1 x 108 colony forming units (CFU) of Brucella abortus strain RB51. Every week for 8 wk beginning 1 wk post inoculation (WPI) 4-6 mice were euthanized via cervical dislocation and necropsied (Cook et al., 2001).
    4 PO RB51 45 in volume female 3-5 month no One hundred eighty captive raised 3–5 mo-old deer mice were randomly divided into four groups of 45 each containing approximately equal numbers of males and nonpregnant females. Mice were given a 2 wk acclimation period prior to beginning the experiment. To allow comparison of oral inoculation (PO) with intraperitoneal inoculation (IP), the standard test method in laboratory mice, both IP and PO experimental groups were established. Because some mortality occurred during the acclimation period, some groups had fewer than 45 mice remaining. The last group of mice (n = 45) was orally dosed with 0.2 ml of 0.85% sterile saline pH 7.0 containing 1 x 108 CFU of RB51. Every week for 8 wk beginning 1 wk post inoculation (WPI) 4-6 mice were euthanized via cervical dislocation and necropsied (Cook et al., 2001).
  • Persistence: All PO inoculated mice cleared the infection by 6 weeks post-inoculation (wpi). While most of the injected mice cleared the infection by 7 wpi, a few required 9 w. With one possible exception (see below), no lesions attributable to brucellosis were noted on gross necropsy. Mice orally inoculated with RB51 developed low level splenic infections with strain RB51, but all were negative by 6 wk PI. RB51 was isolated from the uterus of one orally inoculated mouse necropsied 2 wk PI; no other orally inoculated mouse had a uterine infection. IP inoculated mice had a higher rate of splenic and uterine infections and took longer to clear the infections than orally inoculated mice with some remaining positive at 8 wk PI (Cook et al., 2001).
  • Immune Response: Nine of the 20 (45%) orally inoculated (PO) RB51 mice necropsied in the first 4 wk post-inoculation (WPI) had mild multifocal pleocelluar inflammatory infiltrates in the liver; 3/25 (12%) PO RB51 mice necropsied after 4 WPI had similar lesions. All 10 of the RB51 IP mice necropsied in the first 2 WPI had multifocal pleocellular inflammatory infiltrates in the liver; 2/10 (20%) RB51 IP mice necropsied 3-4 WPI had similar lesions, and only 1 necropsied after 4 WPI from this group had these lesions. Generally, lesions in the IP group were more severe (more cellular infiltrates) than in the PO RB51 group. Lesions were milder in mice at longer times PI. One PO RB51 male mouse necropsied 5 WPI had a mild lymphocytic infiltrate in the interstitium of the seminal vesicle, but no RB51 was recovered from the spleen. Culture results were usually correlated with histologic lesions. Mice with the highest number of colonies of RB51 isolated were those with the most severe lesions (Cook et al., 2001).
  • Side Effects: There were minimal adverse effects attributable to strain RB51. RB51 would not negatively impact P. maniculatus populations if it were used in a field situation. Incidental findings found in some mice in all groups included: mild myocarditis (19% of controls; 30% of RB51 inoculates), mild metritis (10% of controls; 5% of RB51 inoculates), mild multifocal pyogranulomatous pneumonia (8% of controls; 5% of RB51 inoculates), and nematodes in the small intestines and/or cecum (54% of controls; 33% of RB51 inoculates). In a field situation, deer mice would only be exposed to the vaccine PO and not IP. All the orally exposed animals remained clinically healthy with no evidence of illness. Orally exposed mice had fewer CFU cultured from their spleens and uteruses, cleared the vaccine more quickly, and had milder lesions than mice inoculated IP. Strain RB51, with the possible exception of one IP inoculated animal, did not cause significant morbidity or mortality (Cook et al., 2001).

Mouse Response

  • Host Strain: Deer mice (Peromyscus maniculatus)
  • Vaccination Protocol: Deer mice (Peromyscus maniculatus, n = 14) were orally inoculated (PO) with Brucella abortus strain SRB51 or physiologic saline. Oral and rectal swabs and blood samples were collected for bacteriologic evaluation. Rodents were necropsied at 8-10 wk and 12-21 wk post inoculation (WPI). Spleen, liver and reproductive tissues were collected for bacteriologic and histopathologic evaluation (Januszewski et al., 2001).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 RB51 14 in volume female day no 21 mature deer mice of either sex selected from a breeding colony located at the National Wildlife Research Center were
    maintained individually in the Animal Research Building. Deer mice were randomly divided into SRB51-inoculated (n = 14) and control (n = 7) groups. SRB51-inoculated deer mice orally (PO) received SRB51 in a 1 ml volume. Control animals were given the same oral volume of physiologic saline PO. All animals were observed daily. Oral and rectal swabs were collected from all deer mice prior to inoculation and weekly throughout the study. At necropsy, tissues were evaluated for gross lesions and bacteriologic and histologic evaluation (Januszewski et al., 2001).
    2 Saline control 7 in volume female day yes 21 mature deer mice of either sex selected from a breeding colony located at the National Wildlife Research Center were
    maintained individually in the Animal Research Building. Deer mice were randomly divided into SRB51-inoculated (n = 14) and control (n = 7) groups. SRB51-inoculated deer mice orally (PO) received SRB51 in a 1 ml volume. Control animals were given the same oral volume of physiologic saline PO. All animals were observed daily. Oral and rectal swabs were collected from all deer mice prior to inoculation and weekly throughout the study. At necropsy, tissues were evaluated for gross lesions and bacteriologic and histologic evaluation (Januszewski et al., 2001).
  • Persistence: No differences in clinical signs, appetite, weight loss or gain, or activity were observed between saline- and SRB51-inoculated animals. Oral and rectal swabs were negative throughout the study. In tissues obtained from SRB51-inoculated animals, the organism was isolated from 1/6 (17%) deer mice necropsied at 8 WPI. SRB51 was not recovered from deer mice necropsied 12 WPI. SRB51 was not recovered from saline-inoculated deer mice at any time. Results indicate oral exposure to SRB51 does not produce morbibity or mortality in deer mice (Januszewski et al., 2001).
  • Side Effects: One SRB51-inoculated mouse died immediately after inoculation, and one control mouse died at 4 days PI. Deaths were attributed to handling and probable aspiration of the inoculum. Results indicate oral exposure to SRB51 does not produce morbibity or mortality in deer mice (Januszewski et al., 2001).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: Six-week-old BALB/c female mice were inoculated intraperitoneally (IP) with 4 x 108 CFU of Brucella abortus strains RB51 or S2308 or the same volume of saline (negative control). The mice were euthanatized at 6 weeks postimmunization (wpi) by CO2 inhalation. These mice served as splenocyte sources for CTL assays. Splenocytes from strain RB51-infected or saline-inoculated mice were resuspended in 2 ml of c-RPMI and passed through nylon wool columns to enrich for cytotoxic T lymphocytes (CTLs). After 5-day incubation, cells were collected, and live effector cells were obtained by removing dead cells by Histopaque centrifugation. CTLs were also collected and used for flow cytometric analysis, magnetic cell sorting, and colorimetric CTL assay (He et al., 2001).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 RB51 3 in volume female 6 week no Six-week-old BALB/c female mice were inoculated intraperitoneally (IP) with 4 x 108 CFU of Brucella abortus strain RB51. The mice were euthanatized at 6 weeks postimmunization (wpi) by CO2 inhalation. These mice served as splenocyte sources for CTL assays. Splenocytes from strain RB51-infected or saline-inoculated mice were resuspended in 2 ml of c-RPMI and passed through nylon wool columns to enrich for cytotoxic T lymphocytes (CTLs). After 5-day incubation, cells were collected, and live effector cells were obtained by removing dead cells by Histopaque centrifugation. CTLs were also collected and used for flow cytometric analysis, magnetic cell sorting, and colorimetric CTL assay (He et al., 2001).
    2 S2308 3 in volume female 6 week no Six-week-old BALB/c female mice were inoculated IP with 4 x 108 CFU of S2308. The mice were euthanatized at 6 wpi by CO2 inhalation. These mice served as splenocyte sources for CTL assays. Splenocytes from strain RB51-infected or saline-inoculated mice were resuspended in 2 ml of c-RPMI and passed through nylon wool columns to enrich for CTLs. After 5-day incubation, cells were collected, and live effector cells were obtained by removing dead cells by Histopaque centrifugation. CTLs were also collected and used for flow cytometric analysis, magnetic cell sorting, and colorimetric CTL assay (He et al., 2001).
    3 Saline control 3 in volume female 6 week yes Six-week-old BALB/c female mice were inoculated IP with 4 x 108 CFU of RB51 or S2308 or the same volume of saline (negative control). The mice were euthanatized at 6 wpi by CO2 inhalation. These mice served as splenocyte sources for CTL assays. Splenocytes from strain RB51-infected or saline-inoculated mice were resuspended in 2 ml of c-RPMI and passed through nylon wool columns to enrich for CTLs. After 5-day incubation, cells were collected, and live effector cells were obtained by removing dead cells by Histopaque centrifugation. CTLs were also collected and used for flow cytometric analysis, magnetic cell sorting, and colorimetric CTL assay (He et al., 2001).
  • Immune Response: 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. Specifically, Brucella abortus strain RB51 vaccination of mice induced specific cytotoxic T lymphocytes (CTLs) against both RB51- and virulent strain 2308-infected J774.A1 macrophages but not against Listeria monocytogenes-infected J774.A1 cells. The antigen-specific cytotoxic activity was exerted by CTLs but not by NK cells. CD3+ CD4+ T cells secreted the highest level of gamma interferon (IFN) 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-g but demonstrated high levels of specific lysis of Brucella-infected macrophages with no nonspecific lysis (He et al., 2001).
  • Efficacy: RB51 vaccination of mice resulted in the development of a strong cytotoxic T-cell (CTL) response. The specific anti-Brucella cytolytic activity was mainly exerted by CD3+ CD8+ T cells. CD3+ CD4+ T cells also developed after immunization, secreted high levels of IFN, and exhibited certain levels of specific and nonspecific lytic activity against Brucella-infected target cells. NK cells appeared not to contribute significantly to the observed Brucella-specific cytotoxic activity (He et al., 2001).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: This study was designed to determine if a single 0.5 ug administration of recombinant murine interleukin-12 (IL-12) would influence immune responses of mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). A total of 180 (n = 30/treatment) female 10-week-old BALB/c AnNHsD mice were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 5 x 108 cfu of live or gamma-irradiated SRB51 bacteria alone, or in combination with 0.5 ug of IL-12. Control mice received saline +/- 0.5 ug of IL-12 (Lee et al., 1999).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 Saline control 30 in volume female 10 week yes A total of 30 female 10-week-old BALB/c AnNHsD mice were vaccinated IP with 0.2 mL of saline (Lee et al., 1999).
    2 Saline + IL-12 30 in volume female 10 week yes A total of 30 female 10-week-old BALB/c AnNHsD mice were vaccinated intraperitoneally (IP) with 0.2 mL of saline + 0.5 ug of IL-12 (Lee et al., 1999).
    3 Live SRB51 30 in volume female 10 week no A total of 30 female 10-week-old BALB/c AnNHsD mice were vaccinated intraperitoneally (IP) with 0.2 mL of saline containing 5 x 108 cfu of live SRB51 bacteria alone (Lee et al., 1999).
    4 Irradiated SRB51 30 in volume female 10 week no A total of 30 female 10-week-old BALB/c AnNHsD mice were vaccinated IP with 0.2 mL of saline containing 5 x 108 cfu of gamma-irradiated SRB51 bacteria alone (Lee et al., 1999).
    5 Live SRB51 + IL-12 30 in volume female 10 week no A total of 30 female 10-week-old BALB/c AnNHsD mice were vaccinated IP with 0.2 mL of saline containing 5 x 108 cfu of live SRB51 in combination with 0.5 ug of IL-12 (Lee et al., 1999).
    6 Irradiated SRB51 + IL-12 30 in volume female 10 week no A total of 30 female 10-week-old BALB/c AnNHsD mice were vaccinated IP with 0.2 mL of saline containing 5 x 108 cfu of gamma-irradiated SRB51 bacteria in combination with 0.5 ug of IL-12 (Lee et al., 1999).
  • Persistence: Mice vaccinated with live SRB51 or live SRB51+IL-12 had greater serum antibody titers against SRB51 at 2, 4, 8, and 12 wpv when compared to all other treatments. When combined with live or killed SRB51, IL-12 as an adjuvant did not influence serologic responses at 2, 4, 8, or 12 wpv. In the absence of IL-12 as an adjuvant, spleen cells obtained at 12 wk after IP vaccination with live SRB51 had greater stimulation indexes against irradiated S2308 when compared to responses of spleen cells from other treatments. Administration of IL-12 as an adjuvant with live or killed SRB51 did not enhance lymphocyte proliferative responses to S2308 (Lee et al., 1999).
  • Challenge Protocol: Mice (n = 10/treatment) were challenged IP with 2 X 104 cfu of S2308 at 12 weeks post-vaccination (wpv). Blood samples and spleens were obtained from mice in all treatments after CO2/O2 euthanasia at 2, 4, 8, and 12 wpv (n = 5/treatment/time). Following C02/02 euthanasia at 2 weeks after S2308 challenge (wpc), blood and spleens were obtained from mice in all treatments (n = 10/treatment). Cells from 5 mice from each treatment were evaluated separately at 2, 4, 8, and 12 wpv. Following S2308 challenge, spleen cell suspensions from 10 mice from each treatment were combined into 5 separate suspensions (2 mice/suspension, 5 suspensions/treatment) for evaluation of lymphocyte proliferative responses. Cell proliferation results were expressed as stimulation indices (counts/min in presence of antigen divided by counts/min in medium only) (Lee et al., 1999).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 20000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 12 week
  • Efficacy: The clearance of SRB51 from spleens of mice vaccinated with live SRB51 alone, or in combination with IL-12, did not differ at 2, 4, 8, or 12 wk after vaccination. At 12 wk after vaccination, spleens of mice vaccinated with either live SRB51 or live SRB51+IL-12 were culture negative for SRB51. IL-12 did not influence the clearance of strain RB51 from splenic tissue. Spleen weights of mice vaccinated with live SRB51 or live SRB51+IL-12 were greater at 2, 4, 8, or 12 wpv when compared to saline controls, IL-12 controls, or mice receiving either vaccination treatment using killed SRB51. Addition of exogenous IL-12 to culture supernatants of spleen cells obtained at 4 and 8 wk, but not at 2 or 12 wpv, enhanced lymphocyte proliferative responses of all vaccine treatments. Maximal enhancement of lymphocyte response was associated with
    the addition of IL-12. Mice vaccinated with live SRB51 (5 X 108 cfu) had lower spleen weights, total spleen cfu, and spleen cfu/g following challenge at 12 wk after vaccination when compared to mice receiving saline control, IL-12 control, or killed SRB51 treatments. Administration of IL-12 as an adjuvant with live or killed SRB51 did not enhance resistance against experimental challenge with S2308 (Lee et al., 1999).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 2 CFU Colonization of splenic tissues 2 wk following IP challenge with 2 x 104 cfu of S2308 was analyzed via resulting CFU/g spleen +/- IL-12. Mice vaccinated with live SRB51 had lower total spleen cfu and spleen cfu/g (>2 log decreased CFU) following challenge at 12 wk after vaccination when compared to mice receiving saline control, IL-12 control (~0.5 log decreased), or killed (~1 log) SRB51 treatments. Administration of IL-12 as an adjuvant with live or killed SRB51 did not enhance resistance against experimental challenge with S2308 (Lee et al., 1999).
    2 assay of CFU reduction in spleen 0.5 CFU Colonization of splenic tissues 2 wk following IP challenge with 2 x 104 cfu of S2308 was analyzed via resulting CFU/g spleen +/- IL-12. Mice vaccinated with live SRB51 had lower total spleen cfu and spleen cfu/g (>2 log decreased CFU) following challenge at 12 wk after vaccination when compared to mice receiving saline control, IL-12 control (~0.5 log decreased), or killed (~1 log) SRB51 treatments. Administration of IL-12 as an adjuvant with live or killed SRB51 did not enhance resistance against experimental challenge with S2308 (Lee et al., 1999).
    3 assay of CFU reduction in spleen 1 CFU Colonization of splenic tissues 2 wk following IP challenge with 2 x 104 cfu of S2308 was analyzed via resulting CFU/g spleen +/- IL-12. Mice vaccinated with live SRB51 had lower total spleen cfu and spleen cfu/g (>2 log decreased CFU) following challenge at 12 wk after vaccination when compared to mice receiving saline control, IL-12 control (~0.5 log decreased), or killed (~1 log) SRB51 treatments. Administration of IL-12 as an adjuvant with live or killed SRB51 did not enhance resistance against experimental challenge with S2308 (Lee et al., 1999).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following oral (PO) or intraperitoneal (IP) vaccination with strain RB51 (SRB51). Female 10-week-old BALB/c AnNHsD mice were used in the following experiments. Lyophilized SRB51 reconstituted in a 0.15 M NaCl saline solution was used to vaccinate PO by placing 20 ml of saline containing 5 x 108 or 5 x 106 CFU onto the pharyngeal mucosa. Additional groups of mice were vaccinated IP with 0.2 ml of saline containing 5 x 108 or 5 x 106 CFU of SRB51. Nonvaccinated control mice were injected IP with 0.2 ml of saline alone (Stevens et al., 1996).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 PO SRB51 5 x 108 CFU 12 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice were vaccinated PO by placing 20 ml of saline containing 5 x 108 CFU onto the pharyngeal mucosa (Stevens et al., 1996).
    2 PO SRB51 5 x 106 CFU 12 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice were given lyophilized SRB51 reconstituted in a 0.15 M NaCl saline solution PO by placing 20 ml of saline containing 5 x 106 CFU onto the pharyngeal mucosa (Stevens et al., 1996).
    3 IP SRB51 5 x 108 CFU 12 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice were given lyophilized SRB51 reconstituted in a 0.15 M NaCl saline solution was used to vaccinate mice IP with 0.2 ml of saline containing 5 x 108 CFU of SRB51 (Stevens et al., 1996).
    4 IP SRB51 5 x 106 CFU 12 in volume female 10 week no Female 10-week-old BALB/c AnNHsD mice were given lyophilized SRB51 reconstituted in a 0.15 M NaCl saline solution and were vaccinated IP with 0.2 ml of saline containing 5 x 106 CFU of SRB51 (Stevens et al., 1996).
    5 IP Saline control 12 in volume female 10 week yes Female 10-week-old BALB/c AnNHsD mice were injected IP with 0.2 ml of saline alone (Stevens et al., 1996).
  • Persistence: Bacteria persisted in the parotid lymph node for 4 weeks following PO vaccination of mice with 5 x 108 or 5 X 106 CFU of SRB51. Bacteria did not appear in the spleen during 12 weeks after PO vaccination, whereas they did appear in the spleen for 8 weeks following IP vaccination of mice with SRB51. Increased resistance to S2308 infection occurred at 12-20 weeks in mice vaccinated IP with SRB51 but occurred at 12 weeks only in mice vaccinated PO with 5 x 108 CFU SRB51 (Stevens et al., 1996).
  • Immune Response: Oral SRB51 vaccination induced lower levels of antibodies to the surface antigens of intact SRB51 bacteria than did IP vaccination (Stevens et al., 1996).
  • Challenge Protocol: Nonvaccinated control mice and vaccinated mice at 12, 16, or 20 weeks after vaccination were challenged with S2308 by an IP injection of 2 x 104 CFU in 0.2 ml of 0.15 M NaCl saline solution (Stevens et al., 1996).
  • Challenge Detail:
    No. Pathogen Name Dose Route Age Interval
    1 S2308 20000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 12 week
    2 S2308 20000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 16 week
    3 S2308 20000 CFU in volume 0.2 ml Intraperitoneal injection (i.p.) day 20 week
  • Efficacy: 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 PO with SRB51 and challenged with S2308 at 12-20 weeks had lower and less persistent spleen cell proliferation and production of gamma interferon (IFN) in response to S2308 and certain immunodominant S2308 proteins (<18-32 kDa) than did mice vaccinated IP with SRB51. However, mice had similar spleen cell tumor necrosis factor alpha (TNF) production. These results indicate that PO 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 IP vaccination. The lower protective immunity induced by oral vaccination may have resulted from lower and less persistent cell-mediated immunity and IFN production in response to S2308 and S2308 proteins (Stevens et al., 1996).
  • Efficacy Detail:
    No. Efficacy method Result Description Group Efficacy Detail
    1 assay of CFU reduction in spleen 2-3 CFU Increased resistance to S2308 infection, as measured by splenic CFU reduction, occurred at 12-20 weeks in mice vaccinated IP with SRB51 but occurred at 12 weeks only in mice vaccinated PO. Resistance to infection in SRB51-vaccinated mice following challenge infection with S2308. Mice were challenged with S2308 at 12, 16, or 20 weeks after IP or PO vaccination with SRB51. CFU reduction was greatest following IP SRB51 vaccination with 5 x 108 CFU (2-3 log decrease at 12-20 weeks post-challenge) and with 5 x 106 CFU (1.5-2 log decrease). PO SRB51 vaccination led to less of a reduction, whether used at 5 x 108 CFU (0.3-1 log decrease) or at 5 x 106 CFU (0-0.3 log decrease) (Stevens et al., 1996).
    2 assay of CFU reduction in spleen 1.5-2 CFU Increased resistance to S2308 infection, as measured by splenic CFU reduction, occurred at 12-20 weeks in mice vaccinated IP with SRB51 but occurred at 12 weeks only in mice vaccinated PO. Resistance to infection in SRB51-vaccinated mice following challenge infection with S2308. Mice were challenged with S2308 at 12, 16, or 20 weeks after IP or PO vaccination with SRB51. CFU reduction was greatest following 5 x 106 CFU (1.5-2 log decrease). PO SRB51 vaccination led to less of a reduction, whether used at 5 x 108 CFU (0.3-1 log decrease) or at 5 x 106 CFU (0-0.3 log decrease) (Stevens et al., 1996).
    3 assay of CFU reduction in spleen 0.3-1 CFU Increased resistance to S2308 infection, as measured by splenic CFU reduction, occurred at 12-20 weeks in mice vaccinated IP with SRB51 but occurred at 12 weeks only in mice vaccinated PO. Resistance to infection in SRB51-vaccinated mice following challenge infection with S2308. Mice were challenged with S2308 at 12, 16, or 20 weeks after IP or PO vaccination with SRB51. PO SRB51 vaccination led to less of a reduction, whether used at 5 x 108 CFU (0.3-1 log decrease) or at 5 x 106 CFU (0-0.3 log decrease) (Stevens et al., 1996).
    4 assay of CFU reduction in spleen 0-0.3 CFU Increased resistance to S2308 infection, as measured by splenic CFU reduction, occurred at 12-20 weeks in mice vaccinated IP with SRB51 but occurred at 12 weeks only in mice vaccinated PO. Resistance to infection in SRB51-vaccinated mice following challenge infection with S2308. Mice were challenged with S2308 at 12, 16, or 20 weeks after IP or PO vaccination with SRB51. PO SRB51 vaccination led to less of a reduction, just a 0-0.3 log decrease at 5 x 106 CFU (Stevens et al., 1996).

Mouse Response

  • Host Strain: BALB/c
  • Vaccination Protocol: BALB/c mice were inoculated intraperitoneally (IP) with suspensions of Brucella abortus strains 2308 or RB51 or an htrA mutant. Spleens were examined on post-inoculation day (PID) 2, 4, 7, 10, 15, 21, 30, and 60. 132 male BALB/c mice were infected with either one of the 3 B. abortus strains or with saline as a control (Palmer et al., 1996).
  • Vaccination Groups:
    No. Group Name Number of Animals Dose Route Gender Age Control Group? Comment Vaccination Detail
    1 S2308 group 42 in volume male day yes BALB/c mice were inoculated IP with 1 x 104 CFU S2308 in 0.2 ml saline (Palmer et al., 1996).
    2 RB51 group 42 in volume male day no BALB/c mice were inoculated IP with RB51 (5 x 107 CFU) in 0.2 ml saline (Palmer et al., 1996).
    3 htrA group 42 in volume male day no BALB/c mice were inoculated IP with an htrA mutant strain (5 x 104 CFU) in 0.2 ml saline (Palmer et al., 1996).
    4 Saline control 6 in volume male day yes Control BALB/c mice were injected IP with 0.2 ml saline alone (Palmer et al., 1996).
  • Persistence: Brucellae were cultured in high numbers from the spleens of mice infected with strains 2308 or htrA through PID 60; however, mice infected with RB51 cleared the infection between PID 30 and 60. Histopathologic changes in spleens from 2308-infected mice were characterized by marked accumulations of macrophages, which expanded marginal zones beginning as early as PID 7 and persisting through PID 60. Morphometric analysis showed a decrease in splenic white pulp in 2308-infected mice at PID 10, which correlated with the peak of bacterial infection. Although this decrease was significant when compared with values at previous and following time periods, it was not significantly different from white pulp values noted at PID 2 or 4 or the values for control spleens (Palmer et al., 1996).
  • Immune Response: Spleens from RB51-infected mice showed only mild to moderate accumulations of macrophages in marginal zone areas during the peak of RB51 infection (PID 7-10). Morphometric analysis of RB51-infected spleens showed a decrease in white pulp area, which coincided with peak bacterial numbers. However, this decrease was not significant. Spleens from mice infected with htrA showed moderate to marked accumulations of macrophages in marginal zone areas, which persisted through PID 60. Multifocal necrosis in lymphoid follicles as early as PID 4 was seen in both htrA and 2308 infection (Palmer et al., 1996).
  • Efficacy: Morphometric analysis of htrA-infected spleens revealed no significant decrease in white pulp and no obvious correlation with bacterial numbers in the spleen. These results suggest that virulent B. abortus does not induce lymphoid depletion significantly below those values seen in noninfected mice; thus, the possible role of lymphoid depletion in the pathogenesis of brucellosis remains questionable (Palmer et al., 1996).

Mouse Response

Mouse Response

Mouse Response

Mouse Response

Mouse Response

Mouse Response

Mouse Response

Cattle Response

  • Vaccination Protocol: Mature female cattle were immunized with strain 19 ( 2x108 and 6x108 CFU respectively) and 6-12 month old calves with strain 19 ( 4x1010 and 12x1010 CFU ) . Mature females and calves were inoculated with sterile vaccine diluent and formed a non-vaccinated, in-contact control group (Geong et al., 2000).
  • Challenge Protocol: Cattle were allowed to graze for 24 months under conditions where infection was possible (Geong et al., 2000).
  • Efficacy: Of the 599 animals bled initially, 83.5, 72.6, 73.8 and 64.1%, respectively, were represented at 3, 6, 9 and 24 months post infection, respectively. No significant differences between the two villages in the percentage of animals presented at each sampling were found. During the trials, no difference was observed in the presenting of animals identified as seropositive and those identified as seronegative (Geong et al., 2000).

Cattle Response

  • Host Strain: Crossbreed
  • Vaccination Protocol: At Day 0 of the experiment, heifers in the vaccinated group were divided in two sub-groups: 12 heifers were vaccinated at Day 0 of the experiment and the remaining 8 heifers were vaccinated at the 60th day of gestation, with a 2 mL dose. The heifers of the control group received 2 mL of sterile saline solution (Poester et al., 2006).
  • Immune Response: Sera from all animals of RB51 vaccinated and control groups did not show anti-Brucella antibodies on Days −30, 0, 15, and monthly thereafter until the day of challenge (Poester et al., 2006).
  • Challenge Protocol: All animals were challenged with the virulent B. abortus strain 2308. Each heifer received a 3.0 × 107 CFU challenge per heifer (Poester et al., 2006).
  • Efficacy: Vaccination with RB51 vaccine did not result in the production of any antibodies against the O-side chain of lipopolysaccharide. The results indicate that vaccination with RB51 prevented 59.4% of abortions, 58.6% of cow infections, and 61.0% of fetal infections. The relative risk revealed that non-vaccinated animals have 2.462 times higher risk of aborting than RB51-vaccinated animals (Poester et al., 2006).

Cattle Response

  • Vaccination Protocol: Mass vaccination of the cattle population of 3 Azore islands (Terceira, S. Miguel and S. Jorge) targeted breeding herds with the RB51 vaccine supplied by CZ Veterinaria S.A (Pontevedra, Spain). It was administered to heifers or adult female cattle s.c. at 10–34 × 109/dose (2 ml). In herds still infected with an intra-herd incidence of more than 10% after 6 months, all adults were re-vaccinated, as were adjacent herds (Martins et al., 2009).
  • Persistence: During the study period, supramammary (N = 303) and retro-pharyngeal (N = 342) lymph nodes and spleen (N = 298) samples from 343 sero-positive animals slaughtered due to brucellosis suspicion were submitted for culture. Brucella was isolated from 176 animals (51%). Similar isolation rates (39%) were observed for supramammary and retro-pharyngeal lymph nodes, but a lower rate was seen for spleen (7%) (Martins et al., 2009).
  • Immune Response: During the study period, supramammary (N = 303) and retro-pharyngeal (N = 342) lymph nodes and spleen (N = 298) samples from 343 sero-positive animals slaughtered due to brucellosis suspicion were submitted for culture. Brucella was isolated from 176 animals (51%). Similar isolation rates (39%) were observed for supramammary and retro-pharyngeal lymph nodes, but a lower rate was seen for spleen (7%) (Martins et al., 2009).
  • Side Effects: No side-effects, such as abortion were reported (passive reporting). The only outstanding observation that could be related to vaccination was a weak newborn calf from which RB51 was isolated. The annual number of human cases was also low (≤4) and none of them was due to RB51, but to field strain B. abortus biovar 1. It was impossible to relate the disease occurrence in humans with the prevalence in animals (Martins et al., 2009).
  • Challenge Protocol: n/a (Martins et al., 2009)
  • Efficacy: The overall trend is a regular decrease of the three parameters from the beginning of the vaccination programme up to the end of the study period. Average herd incidence, herd prevalence and animal prevalence decreased 69.26%, 39.26% and 75.41% respectively, from 2002 to 2007 for the whole area. Mean within-herd prevalence followed the same pattern, declining on a gradual pace until 2007 (Martins et al., 2009).
  • Description: RB51 vaccine supplied by CZ Veterinaria S.A (Pontevedra, Spain) (Martins et al., 2009).

Cattle Response

  • Vaccination Protocol: To establish the efficacy of RB51 in cattle, female calves were inoculated SC at 3, 5, 7, and 10 months, respectively with RB51 (n = 26), S19 (n = 22), or saline (n = 15) (Cheville, 2000).
  • Persistence: B. abortus strain RB51 was cultured from biopsies of superficial cervical lymph nodes in heifers vaccinated with RB51 and S19 at 10 weeks, but not at 12 weeks (Cheville, 2000).
  • Side Effects: In cattle, RB51 is less virulent than B. abortus cattle vaccine Strain 19.
  • Challenge Protocol: Calves were bred at 16 to 17 months of age and challenged during the first pregnancy with virulent B. abortus (Cheville, 2000).
  • Efficacy: After vaccination, heifers administered RB51 developed no serum antibodies that reacted in the agglutinate test , but yielded a positive dot -blot assay using RB51 antigen . B. abortus strain RB51 was cultured from biopsies of superficial cervical lymph nodes from cows challenged with RB51and S19 vaccines at 10 weeks , but not at 12 weeks. Three month old vaccinated heifers were protected from infection and abortion; strain 19-vaccinated heifers were not infected nor aborted. Control heifers were infected. Vaccination at five and seven months of age gave equivalent, but less than complete protection. Heifers given strain 19 (n = 16) were protected 95%; those given RB51 were 87% protected; controls (administered saline) (n = 15) exhibited a high incidence of infection and abortion. No significant differences in the efficacy of either vaccine was found in animals at any given age. However, when compared to controls marked differences in the efficacy of each vaccine were noted. No gross or microscopic evidence of brucellosis was found in the tissues and organs of cows not infected at birth (Cheville, 2000).

    All heifers vaccinated with RB51 at three months were protected against infection and abortion. Vaccination at five and seven months of age yielded equivalent protection. Heifers given strain 19 were 95% protected. In calves, the results obtained suggest that RB51 protects at dosage levels comparable to those of strain 19.(Cheville, 2000).
  • Description: Strain RB51 is protective in cattle at doses comparable to those of strain 19 (Cheville, 2000). Brucella abortus strain RB51 is the vaccine of choice against brucellosis of cattle in the United States .

Water buffalo Response

  • Vaccination Protocol: Thirty Brucella-free female buffaloes were divided in three groups. Group A consisted of 10 non-pregnant water buffaloes 4–5 years old that were vaccinated once with RB51 (10.2 × 1010 CFU/ml) during lactation. Group B consisted of 10 adult water buffaloes 28–36 months old at the first lactation that were previously vaccinated twice (4 weeks apart) as calves (6–9 months old) with 10.2 × 1010 CFU/ml RB51, but were not revaccinated with RB51 as adults. Group C consisted of 5 adults 4–5 years old during the fourth month of lactation and 5 adult water buffaloes 28–36 months old at the first lactation that were not vaccinated (controls) (Longo et al., 2009).
  • Side Effects: No side effects attributable to vaccine administration were noted in any treatment group. All the experimental water buffaloes appeared clinically normal throughout the study (Longo et al., 2009).
  • Efficacy: RB51 was identified from milk samples collected during the first week post-vaccination (days 1 and 4) in 3/10 of the vaccinated animals of group A but was never isolated in groups B and C. Moreover, RB51 DNA was detected during the first week until the fourth week post-vaccination in the milk samples from group A but was not detected in milk samples of the animals of B and C (Longo et al., 2009).

Squirrel Response

  • Host Strain: Richardson's ground squirrel
  • Vaccination Protocol: Each Richardson’s ground squirrel received 250 µl of inoculum directly into the mouth via tuberculin syringe. All squirrels were observed twice daily for clinical signs for 21 days and once daily thereafter. RB51, strain 19 (S19), and virulent B. abortus strain 9941 (S9941) were administered orally to squirrels to further characterize B. abortus infection in this species. Six groups of nongravid ground squirrels were orally inoculated with 6 x 108 CFU RB51, 2.5 x 104 CFU S19, 2.5 x 107 CFU S19, 1.3 x 106 CFU S9941, 2.1 x 108 CFU S9941, or vaccine diluent (control) (Nol et al., 2009).
  • Persistence: One of 5 animals in the lower-dose S19 group and 2 of 3 animals in the higher-dose S19 group showed persistence of bacteria in various tissues at 14 weeks post-inoculation (WPI). At 18 WPI, 1/5 animals in the RB51 group and 1/5 animals in the high-dose S9941 group were
    culture positive. There was a general lack of persistence of the three strains of B. abortus in Richardson’s ground squirrels at 18 wk PI (Nol et al., 2009).
  • Immune Response: On examination, all livers displayed hepatocellular vacuolization with a pronounced centrilobular pattern consistent with lipidosis and occasional multifocal areas of pleocellular infiltrates consisting of small and large mononuclear cells, suggesting mild hepatitis. One animal in the RB51 group also had focal necrotizing hepatitis. Other lesions observed in these animals included multifocal interstitial pyogranulomatous nephritis and nephrosis, focal necrotizing pancreatitis, and sparse mononuclear cellular infiltrates in the oviduct and uterus (Nol et al., 2009).
  • Side Effects: There was no evidence of pathology caused by B. abortus strains in nonpregnant squirrels based on clinical signs, gross lesions, and microscopic lesions (Nol et al., 2009).
  • Challenge Protocol: Richardson’s ground squirrels experience minimal clinical signs and pathology at challenge levels ranging from 2.5 x 104 CFU to 6 x 108 CFU (Nol et al., 2009).
  • Efficacy: It is unknown if and when the three animals that were culture positive at 14 wk and the two positive animals at 18 wk may have cleared the infection if given more time. No evidence of shedding of RB51 or S19 beyond 2 days PI, and only 2/25 animals cultured at 18 wk showed evidence of persistent infection. Even if infected, squirrels are unlikely to be severely affected by B. abortus or serve as sources of Brucella in the field (Nol et al., 2009).
  • Description: In summary, RB51, S19, and S9941 do not produce disease in nonpregnant Richardson’s ground squirrels when given orally at challenge doses ranging from 2.5 x 104 cfu to 6 x 108 cfu. This species is unlikely to act as a reservoir or source of infection for either vaccine or wild type strains of B. abortus. The Richardson’s ground squirrel would be a poor model for oral Brucella infection as well. These data provide useful information for researchers and those charged with the management of brucellosis in wildlife in the GYA (Nol et al., 2009).

Buffalo Response

  • Host Strain: Bison (Bison bison)
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