Brucella abortus strain RB51 vaccination in elk. II. Failure of high dosage to prevent abortion

Brucella abortus strain RB51 is used as a vaccine because it induces antibodies that do not react on standard serologic tests for brucellosis allowing differentiation between vaccination and infection. Strain RB51 was evaluated in captive elk (Cervus elaphus) to determine if vaccination protected against abortion following experimental challenge. Thirty elk were vaccinated intramuscularly with 1.0 x 10(10) colony-forming units (CFU) of strain RB51 in March 1998. Fourteen of these were given a booster dose of 1.13 x 10(10) CFU exactly 1 yr later. All vaccinated elk seroconverted via a modified dot blot assay to strain RB51 with the booster group having higher titers (P < or = 0.001). Seventeen other elk served as unvaccinated controls. All elk were bred and determined pregnant using pregnancy-specific protein B analysis. Elk were challenged in March 2000 with 1.1 x 10(7) CFU of B. abortus strain 2308 administered intraconjunctivally and all elk seroconverted to strain 2308. Fifteen of 17 control elk aborted; 16 of 16 elk given a single vaccination aborted (P = 0.44); and 13 of 14 elk given a booster aborted (P = 0.86). There were two viable calves in the control group and one in the booster group. Strain 2308 was recovered from fetuses and nonviable calves in all groups. Based on the results of this and other studies, the use of strain RB51 to prevent abortion in elk cannot be recommended.     

Brucella abortus strain RB51 vaccination in elk. I. Efficacy of reduced dosage

Bovine brucellosis is a serious zoonotic disease affecting some populations of Rocky Mountain elk (Cervus elaphus nelsoni) and bison (Bison bison) in the Greater Yellowstone Area, USA. The fear that elk and/or bison may spread Brucella abortus to livestock has prompted efforts to reduce or eliminate the disease in wildlife. Brucella abortus strain RB51 (RB51) vaccine has recently been approved for use in cattle. Unlike strain 19 vaccine, RB51 does not cause false positive reactions on standard brucellosis serologic tests. If effective, it may become the vaccine of choice for wildlife. In February 1995, 45 serologically negative female elk calves were trapped and taken to the Sybille Wildlife Research and Conservation Education Unit near Wheatland, Wyoming, USA. In May 1995, 16 of these elk calves were hand-vaccinated with 1 x 10(9) colony forming units (CFU) of RB51, 16 were vaccinated with 1 x 10(8) CFU RB51 by biobullet, and 13 were given a saline placebo. The elk were bred in fall of 1996 and they were challenged with 1 x 10(7) CFU of B. abortus strain 2308 by intraconjunctival inoculation in March 1997. Thirteen (100%) control elk aborted, 14 (88%) hand-vaccinated elk aborted, and 12 (75%) biobullet vaccinated elk aborted or produced nonviable calves. These results suggest that a single dose of 1 x 10(8) to 1 x 10(9) CFU RB51 does not provide significant protection against B. abortus induced abortion in elk. However, the vaccine appears to be safe at this dose and additional study may reveal a more effective RB51 vaccine regimen for elk.     

Safety of Brucella abortus strain RB51 in bull elk

Some of the elk (Cervus elaphus nelsoni) of the Greater Yellowstone Area (Wyoming, Idaho, Montana; USA) are infected with Brucella abortus, the bacterium that causes bovine brucellosis. Brucella abortus strain RB51 vaccine is being considered as a means to control B. abortus induced abortions in cow elk. However, the most probable vaccination strategies for use in free-ranging elk might also result in some bull elk being inoculated, thus, it is important to insure that the vaccine is safe in these animals. In the winter of 1995, 10 free-ranging bull elk calves were captured, tested for B. abortus antibodies, and intramuscularly inoculated with 1.0 x 10(9) colony forming units (CFU) of B. abortus strain RB51. Blood was collected for hemoculture and serology every 2 wk after inoculation for 14 wk. Beginning 4 mo postinoculation and continuing until 10 mo postinoculation elk were serially euthanized, necropsied, and tissues collected for culture and histopathology. These elk cleared the organism from the blood within 6 wk and from all tissues within 10 mo. No lesions attributable to B. abortus were found grossly and only minimal to mild lymphoplasmacytic epididymitis was found in a few elk on histologic examination. In a separate study, six adult bull elk from Wind Cave National Park (South Dakota, USA) were taken to a ranch near Carrington (North Dakota, USA). Three were orally inoculated with approximately 1.0 x 10(10) CFU of RB51 and three were inoculated with corn syrup and saline. Ninety days post-inoculation semen was examined and cultured from these bulls. Strain RB51 was not cultured from their semen at that time. There were no palpable abnormalities in the genital tract and all elk produced viable sperm. Although they contain small sample sizes, these studies suggest that B. abortus strain RB51 is safe in bull elk.     

Brucella abortus in Bison. II. Evaluation of strain 19 vaccination of pregnant cows

Protection against Brucella abortus induced abortion and infection provided by strain 19 (S19) vaccination was evaluated in American bison (Bison bison). Forty-eight pregnant bison were manually inoculated (MI) with S19 vaccine, 44 were ballistically inoculated (BI) with an absorbable hollow pellet containing lyophilized S19, and 46 were manually injected with buffered saline as non-vaccinated controls (NVC). All bison were Brucella spp. seronegative prior to the experiment, in the second trimester of pregnancy, and were randomly assigned to experimental groups. Approximately 60 days post-vaccination, abortions were observed in the vaccinated bison. Brucella abortus strain 19 was recovered from a bison that had recently aborted, her fetus, and from 11 of 12 other aborted fetuses. Fifty-eight percent (53 of 92) of vaccinated bison aborted, and no abortions were observed in the NVC bison. One cow aborted during her second post-vaccinal pregnancy and S19 was identified from the dam and fetus indicating that chronic S19 infections can occur in bison. Positive antibody titers were present 10 mo post-vaccination in 73% (66 of 91) of the bison. Thirteen mo post-vaccination, 30 MI vaccinates, 27 BI vaccinates, and 30 NVC bison were challenged during the second trimester of pregnancy with 1 x 10(7) CFU of B. abortus strain 2308 via bilateral conjunctival inoculation. Protection against abortion was 67% (P less than or equal to 0.0001) for vaccinated bison compared to 4% in NVC. Protection against B. abortus infection was determined to be 39% (P greater than or equal to 0.001) for vaccinates and 0% (zero of 30) for NCV. Persistent antibody titers, vaccine induced abortions, and chronic S19 infections indicate that the S19 vaccine doses used in this study are not suitable for pregnant bison.     

The role of the vacB gene in the pathogenesis of Brucella abortus

Brucella species are important zoonotic pathogens affecting a wide variety of mammals. Therefore, the identification of new Brucella virulence factors is of great interest in understanding bacterial pathogenesis and immune evasion. In this study, we have identified Brucella abortus vacB gene that presents 2343 nucleotides and 781 amino acids and it shows 39% identity with Shigella flexneri vacB gene that encodes an exoribonuclease RNase R involved in bacterial virulence. Further, we have inactivated Brucella vacB by gene replacement strategy generating a deletion mutant strain. In order to test the role of Brucella vacB in pathogenesis, BALB/c and interferon regulatory factor-1 (IRF-1) knockout (KO) mice received Brucella vacB mutant, the virulent parental strain 2308 or the vaccine strain RB51 and the bacterial CFU numbers in spleens and mous survival were monitored. Our results demonstrated that the B. abortus DeltavacB mutant and the wild type strain 2308 showed similar CFU numbers in BALB/c mice. Additionally, IRF-1 KO mice that received either the vacB mutant or S2308 strain died in 12-14 days postinfection; in contrast, all animals that received the RB51 vaccine strain survived for 30 days postinoculation. In summary, this study reports that the vacB gene in B. abortus has no impact on bacterial pathogenesis.     

Safety of revaccination of pregnant bison with Brucella abortus strain RB51

From December 1998 through February 1999, a study was conducted in a Brucella-infected bison herd to evaluate the safety of booster vaccination of adult bison (Bison bison) with 6 x 10(9) colony forming units (CFU) of Brucella abortus strain RB51 (SRB51) that had previously been vaccinated as yearlings with 1 x 10(10) CFU of SRB51. Abortions or other adverse effects were not observed after SRB51 booster vaccination. At 10 wk after adult vaccination, pregnant and nonpregnant bison (n = 65) were randomly selected for bacteriologic sampling of targeted maternal tissues during abattoir processing. Fetal tissues were also sampled in pregnant bison. The SR351 recovered from tissue samples of eight of 48 pregnant bison and none of 17 nonpregnant bison. In three of the eight culture-positive bison, SRB51 was recovered from fetal tissues. In three additional bison, one pregnant and two nonpregnant, B. abortus biovar 1 field strain was recovered from internal iliac or supramammary lymphatic tissues. Results of this study suggest the possibility that the SRB51 vaccine can be safely used to booster vaccinate pregnant bison in a Brucella-infected bison herd. Our data also reaffirms the potential for B. abortus field strains to persist in bison until attainment of reproductive age, despite extensive use of vaccination and serologic testing.     

Validation of a Brucella abortus competitive enzyme-linked immunosorbent assay for use in Rocky Mountain elk (Cervus elaphus nelsoni)

Brucellosis caused by infection with Brucella abortus is present in some elk (Cervus elaphus nelsoni) of the Greater Yellowstone Area (parts of Wyoming, Montana, and Idaho, USA). Since 1985, the Wyoming Game and Fish Department has vaccinated elk on elk feedgrounds in northwestern Wyoming during the winter months using B. abortus strain 19 (strain 19). Analysis of this vaccination program is hampered by the inability of standard serologic tests to differentiate between strain 19 vaccinated elk and those exposed to field strain B. abortus. In 1993, a competitive enzyme-linked immunosorbent assay (cELISA) was licensed to serologically differentiate between strain 19 vaccinated cattle and cattle exposed to field strain B. abortus. Seven groups of elk sera representing various B. abortus exposure histories were used to validate the cELISA test for elk. The cELISA test differentiated strain 19 vaccinated elk from elk that were challenged with B. abortus strain 2308, a pathogenic laboratory strain. The specificity of the cELISA was 96.8% for elk vaccinated with strain 19 only and sampled between 6 mo and 2 yr post vaccination, or with no B. abortus exposure. The sensitivity of the cELISA was 100%. The cELISA test will be useful in evaluating sera collected from elk in vaccinated, brucellosis endemic herds in the Greater Yellowstone Area.     

An indirect ELISA to detect the serologic response of elk (Cervus elaphus nelsoni) inoculated with Brucella abortus strain RB51

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to identify elk (Cervus elaphus nelsoni) with Brucella abortus strain RB51 (RB51)-specific antibodies using a mouse monoclonal antibody specific for bovine IgG1. This test was relatively easy to perform, accurate, and easily reproducible; therefore it could be standardized for use between laboratories. In addition, we attempted to compensate for inherent variabilities encountered when comparing ELISA readings from multiple samples taken from many animals over time. Optical density (OD) readings for each sample were converted into a percent positivity value for analysis. A negative cutoff value was determined above which a sample was considered to have a significantly elevated anti-RB51 antibody level. Pre- and postvaccination sera from 64 6-8 mo old elk, divided into four groups (females subcutaneously inoculated with saline (control animals), females ballistically inoculated with RB51, females subcutaneously inoculated with RB51, and males subcutaneously inoculated with RB51) were used. All serum samples were collected between 27 April and 15 November 1995. Values for all saline controls were appropriately below the negative cutoff value. All subcutaneously and ballistically inoculated elk were serologically positive to RB51 for at least two sampling periods during the study. The difference in percent positivity values for the ballistically compared to the subcutaneously inoculated groups was not statistically significant at 8, 10, 14, or 18 wk postvaccination. This suggests that processing RB51 into lactose based pellets and ballistically inoculating elk with these pellets does not alter the detectable elk antibody response. Also, inoculated and control animals can be accurately identified with ELISA at 4-8 weeks postvaccination.     

Prevention of vertical transmission of Neospora caninum in C57BL/6 mice vaccinated with Brucella abortus strain RB51 expressing N. caninum protective antigens

Bovine abortions caused by the apicomplexan parasite Neospora caninum have been responsible for severe economic losses to the cattle industry. Infected cows either experience abortion or transmit the parasite transplacentally at a rate of up to 95%. Neospora caninum vaccines that can prevent vertical transmission and ensure disruption in the life cycle of the parasite greatly aid in the management of neosporosis in the cattle industry. Brucella abortus strain RB51, a commercially available vaccine for bovine brucellosis, can also be used as a vector to express plasmid-encoded proteins from other pathogens. Neospora caninum protective antigens MIC1, MIC3, GRA2, GRA6 and SRS2 were expressed in strain RB51. Female C57BL/6 mice were vaccinated with a recombinant strain RB51 expressing N. caninum antigen or irradiated tachyzoites, boosted 4 weeks later and then bred. Antigen-specific IgG, IFN-gamma and IL-10 were detected in vaccinated pregnant mice. Vaccinated mice were challenged with 5 x 10(6)N. caninum tachyzoites between days 11-13 of pregnancy. Brain tissue was collected from pups 3 weeks after birth and examined for the presence of N. caninum by real-time PCR. The RB51-MIC3, RB51-GRA6, irradiated tachyzoite vaccine, pooled strain RB51-Neospora vaccine, RB51-MIC1 and RB51-SRS2 vaccines elicited approximately 6-38% protection against vertical transmission. However, the differences in parasite burden in brain tissue of pups from the control and vaccinated groups were highly significant for all groups. Thus, B. abortus strain RB51 expressing the specific N. caninum antigens induced substantial protection against vertical transmission of N. caninum in mice.     

Caspase-2-Dependent Dendritic Cell Death, Maturation, and Priming of T Cells in Response to Brucella abortus Infection

Smooth virulent Brucella abortus strain 2308 (S2308) causes zoonotic brucellosis in cattle and humans. Rough B. abortus strain RB51, derived from S2308, is a live attenuated cattle vaccine strain licensed in the USA and many other countries. Our previous report indicated that RB51, but not S2308, induces a caspase-2-dependent apoptotic and necrotic macrophage cell death. Dendritic cells (DCs) are professional antigen presenting cells critical for bridging innate and adaptive immune responses. In contrast to Brucella-infected macrophages, here we report that S2308 induced higher levels of apoptotic and necrotic cell death in wild type bone marrow-derived DCs (WT BMDCs) than RB51. The RB51 and S2308-induced BMDC cell death was regulated by caspase-2, indicated by the minimal cell death in RB51 and S2308-infected BMDCs isolated from caspase-2 knockout mice (Casp2KO BMDCs). More S2308 bacteria were taken up by Casp2KO BMDCs than wild type BMDCs. Higher levels of S2308 and RB51 cells were found in infected Casp2KO BMDCs compared to infected WT BMDCs at different time points. RB51-infected wild type BMDCs were mature and activated as shown by significantly up-regulated expression of CD40, CD80, CD86, MHC-I, and MHC-II. RB51 induced the production of cytokines TNF-α, IL-6, IFN-γ and IL12/IL23p40 in infected BMDCs. RB51-infected WT BMDCs also stimulated the proliferation of CD4(+) and CD8(+) T cells compared to uninfected WT BMDCs. However, the maturation, activation, and cytokine secretion are significantly impaired in Casp2KO BMDCs infected with RB51 or Salmonella (control). S2308-infected WT and Casp2KO BMDCs were not activated and could not induce cytokine production. These results demonstrated that virulent smooth strain S2308 induced more apoptotic and necrotic dendritic cell death than live attenuated rough vaccine strain RB51; however, RB51, but not its parent strain S2308, induced caspase-2-mediated DC maturation, cytokine production, antigen presentation, and T cell priming.     

Brucella abortus in captive bison. I. Serology, bacteriology, pathogenesis, and transmission to cattle

Two groups of six, non-brucellosis vaccinated, brucellosis seronegative pregnant American bison (Bison bison) were individually challenged with 1 x 10(7) colony forming units (CFU) of Brucella abortus strain 2308. Three days after challenge, each bison group was placed in a common paddock with six non-vaccinated, brucellosis susceptible, pregnant domestic heifers. In a parallel study, two groups of six susceptible, pregnant cattle were simultaneously challenged with the identical dose as the bison and each group was placed with six susceptible cattle in order to compare bison to cattle transmission to that observed in cattle to cattle transmission. Blood samples were collected from bison and cattle weekly for at least 1 mo prior to exposure to B. abortus and for 180 days post-exposure (PE). Sera from the bison and cattle were evaluated by the Card, rivanol precipitation, standard plate agglutination, standard tube agglutination, cold complement fixation tube, warm complement fixation tube, buffered acidified plate antigen, rapid screening, bovine conjugated enzyme linked immunosorbent assay, bison or bovine conjugated enzyme linked immunosorbent assay, and the hemolysis-in-gel techniques for the presence of antibodies to Brucella spp. At the termination of pregnancy by abortion or birth of a live-calf, quarter milk samples, vaginal swabs, and placenta were collected from the dam. Rectal swabs were collected from live calves, and mediastinal lymph nodes, abomasal contents and lung were taken at necropsy from aborted fetuses for culture of Brucella spp. These tissues and swabs were cultured on restrictive media for the isolation and identification of Brucella spp. Pathogenesis of brucellosis in bison was studied in an additional group of six pregnant bison which were challenged with 1 x 10(7) CFU of B. abortus strain 2308. One animal was euthanatized each week PE. Tissues were collected at necropsy and later examined bacteriologically and histologically. Lesions of brucellosis in bison did not significantly differ grossly or histologically from those in cattle. There were six abortions and two nonviable calves in the bison group, as compared to nine abortions in the 12 similarly inoculated cattle. As determined by bacterial isolations, transmission of B. abortus from bison to cattle (five of 12 susceptible cattle became infected) did not differ statistically from cattle to cattle transmission (six of 12 susceptible cattle became infected) under identical conditions. No single serologic test was constantly reliable to diagnosing B. abortus infected bison for 8 wk PE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prevention of lethal experimental infection of C57BL/6 mice by vaccination with Brucella abortus strain RB51 expressing Neospora caninum antigens

Bovine abortions caused by the intracellular protozoal parasite Neospora caninum are a major concern to cattle industries worldwide. A strong Th1 immune response is required for protection against N. caninum. Brucella abortus strain RB51 is currently used as a live, attenuated vaccine against bovine brucellosis. Strain RB51 can also be used as an expression vector for heterologous protein expression. In this study, putative protective antigens of N. caninum MIC1, MIC3, GRA2, GRA6 and SRS2, were expressed individually in B. abortus strain RB51. The ability of each of the recombinant RB51 strains to induce N. caninum-specific immunity was assessed in C57BL/6 mice. Mice were immunised by two i.p. inoculations, 4 weeks apart. Five weeks after the second immunisation, spleen cells from the vaccinated mice secreted high levels of IFN-gamma and IL-10 upon in vitro stimulation with N. caninum whole cell lysate antigens. N. caninum-specific antibodies of both IgG1 and IgG2a subtypes were detected in the serum of the vaccinated mice. Mice in the vaccinated and control groups were challenged with 2 x 10(7)N. caninum tachyzoites i.p. and observed for 28 days after vaccination. All unvaccinated control mice died within 7 days. Mice in the MIC1 and GRA6 vaccine groups were completely protected while the mice in the SRS2, GRA2 and MIC3 vaccinated groups were partially protected and experienced 10-50% mortality. The non-recombinant RB51 vector control group experienced an average protection of 69%. These results suggest that expression of protective antigens of N. caninum in B. abortus strain RB51 is a novel approach towards the development of a multivalent vaccine against brucellosis and neosporosis.     

Safety of Brucella abortus strain RB51 vaccine in non-target ungulates and coyotes

Brucellosis is endemic in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area (GYA; USA). It is possible that an oral brucellosis vaccine could be developed and disseminated in the GYA to reduce disease transmission. Should this occur, non-target species other than elk and bison may come in contact with the vaccine resulting in morbidity or mortality. To assess biosafety, bighorn sheep (Ovis canadensis; n = 10), pronghorn (Antilocapra americana; n = 9), mule deer (Odocoileus hemionus; n = 11), moose (Alces alces shirasi; n = 10), and coyotes (Canis latrans; n = 24) were given a single oral dose of at least 1.0 x 10(10) colony-forming units of Brucella abortus strain RB51 vaccine (RB51). Animals were randomly divided into vaccinated and control groups. Ungulates were captured, blood sampled, and swabs taken from the nares, rectum, and vagina for bacterial culture on day 0, 42, and 84 post-inoculation (PI). On day 42, the vaccinated group became a control group and vice versa in a crossover design. Blood and swab samples were taken from coyotes on days 0, 14, 28, and 42 PI. There was no crossover for the coyote study. Two coyotes from each group were also euthanized and cultured for RB51 on days 42, 84, 168, and 336 PI. Blood samples were analyzed for hematologic changes and antibodies to RB51 using a modified dot-blot assay. No morbidity or mortality as a result of vaccination was observed in any animal. There were no differences in hematologic parameters at any time for ungulate species; vaccinated coyotes had higher hematocrit, hemoglobin, and eosinophil counts (P < or = 0.006). All individuals, except some moose, seroconverted to RB51. Strain RB51 was cultured from oropharyngeal lymph nodes from one coyote 42 days PI and from a moose 117 days PI. This study suggested that a single oral dose of RB51 was safe in these species.     

Safety of Brucella abortus strain RB51 in deer mice

Brucella abortus strain RB51 is an approved brucellosis vaccine for use in cattle that may have potential as an oral vaccine for use in elk (Cervus elaphus) and/or bison (Bison bison). 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. In February 1994, 90 mice were orally dosed or intraperitoneally injected with 1 x 10(8) colony forming units strain RB51 and 77 controls were similarly dosed with sterile saline. At weekly intervals through early April 1994, 4 to 6 mice from each group were euthanized, gross necropsies performed, spleens and uteruses cultured, and tissues examined histologically. All orally inoculated mice cleared the infection by 6 wk post-inoculation (PI). While most of the injected mice cleared the infection by 7 wk PI, a few required 9 wk. There were minimal adverse effects attributable to strain RB51. Apparently, strain RB51 would not negatively impact P. maniculatus populations if it were used in a field situation. Also, deer mice appear to be able to clear the vaccine in 6 to 9 wk, thus the probability of these mice transmitting the vaccine to other animals is low.     

Immune responses of elk to vaccination with Brucella abortus strain RB51

In a study conducted from January to August 2000, elk (Cervus elaphus) were vaccinated with Brucella abortus strain RB51 (SRB51, n = 6) or injected with 0.15 M NaCl solution (n = 3) at approximately 6 mo of age. Beginning at 2 wk and continuing to 25 wk after vaccination, SRB51-vaccinated elk had greater antibody responses (P < 0.05) to SRB51 when compared to nonvaccinated elk. Peripheral blood mononuclear cells (PBMC) from SRB51-vaccinated elk had greater (P < 0.05) proliferative responses to SRB51 at 18 wk after vaccination when compared to responses of nonvaccinated elk. Strain RB51 was recovered from blood samples of all vaccinates at 2 wk, and three of six vaccinates at 4 wk after vaccination. The SRB51 vaccine strain was recovered from the superficial cervical lymph node of all vaccinates sampled at 6 wk after vaccination. but not from lymph node samples obtained from vaccinates at 12 or 18 wk after vaccination. At 34 wk after vaccination, SRB51 was recovered from the bronchial lymph node of one of five vaccinates but not from other tissues. Strain RB51 was not recovered at any time from samples obtained from nonvaccinated elk. This study suggests that following vaccination with SRB51, elk remain bacteremic for a prolonged period of time, rapidly develop high antibody titers, and are slower to develop detectable proliferative responses in PBMC when compared to responses of cattle or bison (Bison bison).     

Effect of Brucella abortus transfer factor in preventing murine brucellosis

Abstract Mice vaccinated with a protein extract of attenuated Brucella abortus strain 19 had increased resistance to infection with virulent B. abortus strain 2308 and had increased antibody responses to strain 2308. However, resistance to infection and antibody responses were not increased when nonvaccinated recipient mice were given transfer factor preparations that were obtained from either vaccinated donor mice or strain 2308-infected donor mice. Vaccination of mice with the strain 19 extract plus treatment with each transfer factor preparation also did not further increase resistance to infection or antibody responses when compared with mice that received the vaccine alone. These results suggest that transfer factor from mice that have either vaccine-induced protective immunity to B. abortus or active B. abortus infections does not enhance antibody responses and resistance to infection with B. abortus .     

Safety and immunogenicity of Brucella abortus strain RB51 vaccine in cross bred cattle calves in India

Safety and immunogenicity of Brucella abortus RB51 vaccine has been evaluated in an organised dairy farm in India. All the cattle (r = 29) vaccinated with strain RB51 'responded' to the vaccine as demonstrated by iELISA using acetone killed strain RB51 antigen. The percentage responders at day 35, 60 and 90 post vaccination were 100%, 95% and 20%, respectively. Strain RB51 was able to elicit a good IFN-gamma response from vaccinated animals. The post-vaccination time point analysis indicated that the cumulative IFN-gamma response of whole blood from vaccinates stimulated with heat killed RB51 antigen was elicited in 80% of calves at 60 days post vaccination. Absence of strain RB51 in the secretions and excretion and lack of local or systemic reaction indicated the safety of the vaccine.     

Immune responses of bison to ballistic or hand vaccination with Brucella abortus strain RB51

From January through July of 2000, a study was conducted to evaluate clearance, immunologic responses, and potential shedding of Brucella abortus strain RB51 (SRB51) following ballistic or subcutaneous (SQ) vaccination of 7 mo old bison (Bison bison) calves. Ten bison calves were vaccinated SQ with 1.4 x 10(10) colony-forming units (CFU) of SRB51 and five calves were inoculated SQ with sterile 0.15 M sodium chloride. An additional 10 bison calves were ballistically inoculated in the rear leg musculature with 1 x 10(10) CFU of SRB51 and five calves were ballistically inoculated with an empty Biobullet. Serologic responses were monitored at 0, 2, 4, 6, 8, 12, 18, and 24 wk using the standard tube agglutination test and a dot-blot assay. Swabs from rectal, vaginal, nasal, and ocular mucosal surfaces, and blood were obtained for culture from all bison at 2, 4, 6, and 8 weeks post-inoculation to evaluate potential shedding by vaccinated bison or persistent septicemia. The superficial cervical lymph node was biopsied in eight ballistic and eight hand vaccinated bison at 6 or 12 wk to evaluate clearance of the vaccine strain from lymphatic tissues. Lymphocyte proliferative responses to irradiated SRB51 bacteria were evaluated in peripheral blood mononuclear cells (PBMC) at 4, 6, 8, 12, 18, and 24 wk after inoculation. Serum obtained from hand or ballistically vaccinated bison demonstrated antibody responses on the dot-blot assay that were greater than control bison (saline or empty Biobullet) at 2, 4, 6, and 8 wk after vaccination. Antibody titers of ballistically vaccinated bison did not differ (P > 0.05) from hand vaccinated bison at any sampling time. Blood samples obtained from all bison at 2, 4, 6 and 8 wk after vaccination were negative for SRB51. One colony of SRB51 was recovered from the vaginal swab of one ballistically vaccinated bison at 2 wk after vaccination. All other ocular, vaginal, nasal, and rectal swabs were culture negative for SRB51. Strain RB51 was recovered from superficial cervical lymph nodes of hand and ballistic vaccinated bison at 6 (two of four and two of four bison, respectively) and 12 wk (three of four and one of four bison, respectively). Serologic tests and bacterial culture techniques failed to demonstrate infection of nonvaccinated bison. Peripheral blood mononuclear cells obtained from hand vaccinated bison had greater (P < 0.05) proliferative responses to strain RB51 bacteria when compared to PBMC from nonvaccinated and ballistically vaccinated bison. Proliferative responses of PBMC from ballistically vaccinated bison did not differ (P > 0.05) at any sampling time from proliferative responses of PBMC from control bison. Serum alpha 1-acid glycoprotein concentrations, plasma fibrinogen, and total protein concentrations were not influenced by treatments. Ballistic delivery of SRB51 did not induce adverse effects or influence clearance of the vaccine strain. There were no proliferative responses of PBMC to SRB51 in bison ballistically vaccinated with SRB51; whereas bison inoculated with SRB51 by hand injection had greater proliferative responses than control or ballistically vaccinated bison. Our study suggests that ballistic delivery may require a greater dose of SRB51 to induce cell-mediated immune responses in bison that are comparable to those induced by hand injection, and that ballistic or hand delivery of 1 x 10(10) CFU of SRB51 is safe in bison calves.     

Antigenic, immunologic and genetic characterization of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B. abortus RB51, B. melitensis B115 and B. melitensis B18. RB51 derived from B. abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B. melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.     

A potent Brucella abortus 2308 Δery live vaccine allows for the differentiation between natural and vaccinated infection

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. However, the current Brucella abortus vaccines (S19 and RB51) are deficient; they can cause abortion in pregnant animals. Moreover, when the vaccine S19 is used, tests cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent vaccine is needed. A Brucella abortus 2308 ery promoter mutant (Δery) was constructed to overcome these drawbacks. The growth of the Δery mutant was significantly attenuated in macrophages and mice and induced high protective immunity in mice. Moreover, Δery induced an anti-Brucella-specific IgG (immunoglobulin G) response and stimulated the expression of interferon-gamma (INF-γ) and interleukin-4 (IL-4). Furthermore, the expression of EryA antigen allowed for the serological differentiation between natural and vaccinated infection in mice. These results indicate that the Δery mutant is a potential attenuated live vaccine candidate against virulent Brucella abortus 2308 (S2308) infection.