Enhanced efficacy of recombinant Brucella abortus RB51 vaccines against B. melitensis infection in mice

Brucella abortus strain RB51 is an attenuated rough strain, currently being used as the official live vaccine for bovine brucellosis in the USA and several other countries. In strain RB51, the wboA gene, encoding a glycosyltransferase required for the O-side chain synthesis, is disrupted by an IS711 element. Recently, we have demonstrated that strain RB51WboA, RB51 complemented with a functional wboA gene, remains rough but expresses low quantities of O-side chain in the cytoplasm. Mice vaccinated with strain RB51WboA develop greatly enhanced resistance against challenge with B. abortus virulent strain 2308. We have also demonstrated that overexpression of Cu/Zn superoxide dismutase (SOD) in strain RB51 (RB51SOD) significantly increases its vaccine efficacy against strain 2308 challenge. In this study, we constructed a new recombinant strain, RB51SOD/WboA, that over expresses SOD with simultaneous expression of O-side chain in the cytoplasm. We tested the vaccine potential of strains RB51SOD, RB51WboA, RB51SOD/WboA against challenge with virulent Brucella melitensis 16M and B. abortus 2308 in mice. In comparison with strain RB51, strain RB51SOD induced better protection against strain 2308, but not strain 16M, challenge. Similar to strain RB51WboA, vaccination with strain RB51SOD/WboA resulted in complete protection of the mice from infection with strain 2308. When challenged with strain 16M, mice vaccinated with either strain RB51WboA or strain RB51SOD/WboA were significantly better protected than those vaccinated with strain RB51 or RB51SOD. These results suggest that strains RB51WboA and RB51SOD/WboA are good vaccine candidates for inducing enhanced protection against B. melitensis infection.     

Use of polymerase chain reaction to identify Brucella abortus strain RB51 among Brucella field isolates from cattle in Italy

Brucella abortus strain RB51, a rough mutant of the B. abortus 2308 virulent strain, was recently approved in the United States as the official vaccine for brucellosis in cattle. Following recent evidence of unauthorized use of RB51 vaccine in Italy, where the use of vaccines for brucellosis is no longer allowed, the suitability of an RB51-specific polymerase chain reaction assay for identifying the RB51 strain among Brucella field isolates from cattle in Italy was investigated. The oligonucleotide primers used in this study, belonging to a six-primer cocktail for Brucella species previously described by other authors, allowed the amplification of a 364-base pair (bp) fragment specific for RB51 and its parent strain 2308, and a 498-bp product specific for B. abortus. In addition, unresolved bands ranging from 600 to 700 bp were observed from RB51 strain. Brucella abortus biovars 1, 2 and 4 have only one specific sensitive 498-bp band. The B. abortus biovars 3, 5 and 6 did not give any signal. The 498-bp product from a reference Brucella strain was sequenced and submitted to EMBL with the accession number AJ271969 while the 364-bp fragment from RB51 strain was submitted to EMBL database with accession number AJ271968. The sequence studies confirmed the specificity of the detected fragments. No amplification was obtained by testing DNA from strains antigenically related to Brucella, such as Yersinia enterocolitica O:9, Escherichia coli O:157, Salmonella urbana and Pasteurella multocida. The results of this study indicate that this technique, in combination with specific serological tests, could be a useful diagnostic method to verify the use of RB51 vaccine and can contribute to the creation of a databank of circulating strains.     

Comparative behaviour of Brucella abortus strains 19 and RB51 in the pregnant mouse.

Pregnant BALB/c mice received various doses of either Brucella abortus strain 19, a smooth vaccine strain, or B abortus strain RB51, a stable rough organism, intraperitoneally on day 9 of gestation to compare the relative pathogenicity of the two attenuated strains. Nine days after inoculation, spleens and placentas were collected for bacteriological and histopathological examination. A dose of 10(7.5) and strain 19 organisms produced a severe necrosuppurative placentitis occasionally accompanied by fetal death. This dose resulted in a 10-fold higher level of splenic infection than did a dose of 10(9.5) strain RB51 organisms, which produced only mild to minimal placentitis not associated with fetal death. Strain 19 infected mice showed seroconversion in the standard tube agglutination test in contrast to the seronegative titre of strain RB51 infected mice. The results of this study corroborate previous investigations on the relative pathogenicity and the serological response of the non-pregnant mouse to strain RB51.     

Comparison between immune responses and resistance induced in BALB/c mice vaccinated with RB51 and Rev. 1 vaccines and challenged with Brucella melitensis bv. 3

BALB/c mice were immunized with live rough Brucella abortus RB51 or smooth Brucella melitensis Rev. 1 vaccines and challenged with a B. melitensis field strain. 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, namely; 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. However, RB51 vaccine has the advantage that it would not elicit antibodies to standard serological tests based on the LPS O antigen. RB51 vaccine could therefore be used for control of B. melitensis infection and avoid confusion in the use of standard sero-diagnostic tests.     

Reduced cerebral infection of Neospora caninum in BALB/c mice vaccinated with recombinant Brucella abortus RB51 strains expressing N. caninum SRS2 and GRA7 proteins

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of bovine neosporosis, an important disease affecting the reproductive performance of cattle worldwide. Currently there is no effective vaccine available to prevent N. caninum infection in cattle. In this study, we examined the feasibility of developing a live, recombinant N. caninum vaccine using Brucella abortus vaccine strain RB51 as the expression and delivery vector. We generated two recombinant RB51 strains each expressing SRS2 (RB51/SRS2) or GRA7 (RB51/GRA7) antigens of N. caninum. BALB/c mice immunized by single intraperitoneal inoculation of the recombinant RB51 strains developed IgG antibodies specific to the respective N. caninum antigen. In vitro stimulation of splenocytes from the vaccinated mice with specific antigen resulted in the production of interferon-gamma, but not IL-5 or IL-10, suggesting the development of a Th1 type immune response. Upon challenge with N. caninum tachyzoites, mice vaccinated with strain RB51/SRS2, but not RB51/GRA7, showed significant resistance to cerebral infection when compared to the RB51 vaccinated mice, as determined by the tissue parasite load using a real-time quantitative TaqMan assay. Interestingly, mice vaccinated with either strain RB51 or RB51/GRA7 also contained significantly lower parasite burden in their brains compared to those inoculated with saline. Mice vaccinated with strain RB51/SRS2 or RB51/GRA7 were protected to the same extent as the strain RB51 vaccinated mice against challenge with B. abortus virulent strain 2308. These results suggest that a recombinant RB51 strain expressing an appropriate protective antigen(s), such as SRS2 of N. caninum, can confer protection against both neosporosis and brucellosis.     

Intracellular trafficking study of a RB51 B. abortus vaccinal strain isolated from cow milk

Brucella is responsible for one of the major worldwide zoonoses. Over the last century, several vaccines have been used against brucellosis. Among these, the rough vaccine Brucella abortus RB51 was introduced with the idea that it would not interfere with the diagnosis of brucellosis. Recently, RB51 has been isolated from milk and vaginal exudates from vaccinated cows, thus raising the possibility of extensive bacterial replication in these animals. We hypothesized that shedding of RB51 might be related to a change in its intracellular cell cycle. Therefore, we have compared the intracellular trafficking in CHO cells of the virulent B. abortus 2308 and two RB51 strains, the vaccinal strain and the one isolated from cow milk. Both RB51 strains were transiently observed in phagosomes characterized by the presence of the early endosomal marker EEA1 and then were found in cathepsin D-enriched lysosomal compartments, in which they eventually underwent degradation at later post-infection times. In contrast, the virulent 2308 strain replicated within the endoplasmic reticulum. These results suggest that a change in intracellular trafficking cannot account for Brucella shedding in adult vaccinated cows.     

Genomic fingerprinting and development of a dendrogram for Brucella spp. isolated from seals, porpoises, and dolphins.

Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species.     

Live Brucella abortus rough vaccine strain RB51 stimulates enhanced innate immune response in vitro compared to rough vaccine strain RB51SOD and virulent smooth strain 2308 in murine bone marrow-derived dendritic cells

Brucella spp. are Gram-negative, coccobacillary, facultative intracellular pathogens. B. abortus strain 2308 is a pathogenic strain affecting cattle and humans. Rough B. abortus strain RB51, which lacks the O-side chain of lipopolysaccharide (LPS), is the live attenuated USDA approved vaccine for cattle in the United States. Strain RB51SOD, which overexpresses Cu–Zn superoxide dismutase (SOD), has been shown to confer better protection than strain RB51 in a murine model. Protection against brucellosis is mediated by a strong CD4+ Th₁ and CD8+ Tc₁ adaptive immune response. In order to stimulate a robust adaptive response, a solid innate immune response, including that mediated by dendritic cells, is essential. As dendritic cells (DCs) are highly susceptible to Brucella infection, it is possible that pathogenic strains could limit the innate and thereby adaptive immune response. By contrast, vaccine strains could limit or bolster the innate and subsequent adaptive immune response. Identifying how Brucella vaccines stimulate innate and adaptive immunity is critical for enhancing vaccine efficacy. The ability of rough vaccine strains RB51 and RB51SOD to stimulate DC function has not been characterized. We report that live rough vaccine strain RB51 induced significantly better (p ≤ 0.05) DC maturation and function compared to either strain RB51SOD or smooth virulent strain 2308, based on costimulatory marker expression and cytokine production.     

Diagnostic characterization of a feral swine herd enzootically infected with Brucella.

Eighty feral swine were trapped from a herd that had been documented to be seropositive for Brucella and which had been used for Brucella abortus RB51 vaccine trials on a 7,100-hectare tract of land in South Carolina. The animals were euthanized and complete necropsies were performed. Samples were taken for histopathology, Brucella culture, and Brucella serology. Brucella was cultured from 62 (77.5%) animals. Brucella suis was isolated from 55 animals (68.8%), and all isolates were biovar 1. Brucella abortus was isolated from 28 animals (35.0%), and isolates included field strain biovar 1 (21 animals; 26.3%), vaccine strain Brucella abortus S19 (8 animals, 10.0%), and vaccine strain Brucella abortus RB51 (6 animals, 7.5%). Males were significantly more likely to be culture positive than females (92.9% vs. 60.6%). Thirty-nine animals (48.8%) were seropositive. Males also had a significantly higher seropositivity rate than females (61.9% vs. 34.2%). The relative sensitivity rates were significantly higher for the standard tube test (44.6%) and fluorescence polarization assay (42.6%) than the card agglutination test (13.1%). Lesions consistent with Brucella infection were commonly found in the animals surveyed and included inflammatory lesions of the lymph nodes, liver, kidney, and male reproductive organs, which ranged from lymphoplasmacytic to pyogranulomatous with necrosis. This is the first report of an apparent enzootic Brucella abortus infection in a feral swine herd suggesting that feral swine may serve as a reservoir of infection for Brucella abortus as well as Brucella suis for domestic livestock.     

Oral vaccination of sexually mature pigs with Brucella abortus vaccine strain RB51

OBJECTIVE: To develop a novel oral vaccine delivery system for swine, using the rough vaccine strain of Brucella abortus. ANIMALS: 56 crossbred pigs from a brucellosis-free facility. PROCEDURE: In 3 separate experiments, pigs were orally vaccinated with doses of 1 x 10(9) to > 1 x 10(11) CFU of strain RB51 vaccine. The vaccine was placed directly on the normal corn ration, placed inside a whole pecan, or mixed with cracked pecans and corn. RESULTS: Oral vaccination of pigs with vaccine strain RB51 resulted in a humoral immune response to strain RB51 and short-term colonization of the regional lymph nodes. CONCLUSIONS AND CLINICAL RELEVANCE: A viscous liquid such as Karo corn syrup in association with pecans that scarify the oral mucosa are necessary when placing the live vaccine directly onto corn or other food rations. Doses of > 1 x 10(11) CFU of RB51 organisms/pig in this mixture ensures 100% colonization of regional lymph nodes via the oral route. This method may allow an efficient and economical means to vaccinate feral swine for brucellosis.     

Biological properties of RB51; a stable rough strain of Brucella abortus

A rifampin-resistant mutant of Brucella abortus, designated RB51, was derived by repeated passage of strain 2308 on Trypticase soy supplemented with 1.5% agar and varying concentrations rifampin or penicillin. The RB51 colonies absorbed crystal violet and RB51 cell suspensions autoagglutinated, indicating a rough type colonial morphology for this strain. No O-chain component was detected in lipopolysaccharide (LPS) extracted from RB51 on SDS-PAGE gels stained with silver. Western blot analysis with the monoclonal antibody BRU 38, which is specific for the perosamine homopolymer O-chain of smooth Brucella LPS, indicated that the LPS of RB51 is highly deficient in O-chain when compared with the parenteral smooth strain 2308 or rough strain 45/20. Biochemically, RB51 resembles parental strain 2308 in its ability to utilize erythritol. Intraperitoneal inoculation 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. Mice infected with RB51 produce antibodies against B. abortus antigens including class 2 and 3 outer membrane proteins but not against the O-chain. Furthermore, rabbits, goats and cattle hyperimmunized with sonicates of RB51 develop antibodies to B. abortus cellular antigens but do not develop antibodies specific for the O-chain. Immunization of mice with 1 x 10(8) viable RB51 organisms confers significant protection against challenge with virulent B. abortus strain 2308.     

Bacterial survival, lymph node changes, and immunologic responses of cattle vaccinated with standard and mutant strains of Brucella abortus.

Forty-eight cattle were used in 4 experiments; 6-week-old calves in experiments 1-3 (n = 24) and 10-month-old heifers in experiment 4 (n = 24). In experiments 1-3, 7 groups of 3 calves each were inoculated SC with 5 strains of Brucella abortus: virulent strain 2308 (2 groups), vaccine strain 19 (2 groups), and mutant strains RB51. 19 delta 31K, and 19 delta SOD. Sera and lymph node tissues were examined at 2-week intervals for evidence of infection. At postinoculation (PI) week 12, 2 calves in each group were given dexamethasone for 5 days. Calves were then euthanatized and lymphoid tissue, spleen, liver, and bone marrow were examined for evidence of B abortus. Calves given strain 2308 had large numbers of bacteria in their lymph nodes, marked granulomatous lymphadenitis in the deep cortex, and loss of lymphoid cells in superficial cortical areas. In addition, they had high serum antibody titers at PI week 16. Calves given strain 19, or genetic mutants derived from strain 19, cleared bacteria from lymph nodes more rapidly, had less lymphoid destruction, and developed antibody titers that did not persist for 16 weeks. The RB51 strain (rough) was cleared most rapidly from lymphoid tissues and induced serum antibody responses only to the core of the lipopolysaccharide molecule. Treatment of calves with dexamethasone did not cause B abortus to reappear in tissues of any calves, nor did serum antibody titers increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serologic responses, biosafety and clearance of four dosages of Brucella abortus strain RB51 in 6-10 months old water buffalo (Bubalus bubalis)

Thirty water buffalo were obtained from a brucellosis-free farm in order to evaluate antibody responses, bacterial clearance and safety to Brucella abortus strain RB51 vaccine in a dose response study. The animals were randomly divided into five treatment groups. Groups I-V received the recommended dose of RB51 vaccine (RD) once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart and saline once, respectively. Antibody responses to RB51 were monitored at 2, 4, 6, 8, 10, 12, 16 18, 22, 24 and 27 post-initial-inoculation weeks (PIW). Clearance of RB51 from the prescapular lymph node was evaluated at 2, 4, 6, 12, 18 and 24 PIW for groups 1, III and V and at 6, 8, 10, 16, 22 and 27 PIW for groups II and IV. To evaluate shedding of the RB51 strain, nasal, conjunctival, vaginal or preputial swabs were taken from all experimental animals at 1, 2, 3, 4, 6, 8 and 12 PIW. Sera taken at all PIW were negative for field strain B. abortus by both the buffered plate agglutination test (BPAT) and competitive enzyme-linked immunosorbent assay (c-ELISA). Antibody responses to RB51 were demonstrated in all vaccinates but not in the controls, up to 12 PIW, by complement fixation test (CFT) and the dot-blot assay with an 83.7% agreement for both tests. Clearance of RB51 occurred between 6 and 12 PIW in group I but less than 2 weeks after booster vaccinations in groups II and IV and between 4 and 6 PIW in group III. RB51 was not recovered at any time from swabs obtained from either RB51-vaccinates or non-vaccinates. The results of this study indicate that serologic responses to RB51 vaccination can be monitored by both CFT and dot-blot assay in water buffalo. Our data also indicates that RB51 vaccination does not interfere with brucellosis sero-surveillance and is safe (no serological and bacteriological evidence of spread to non-vaccinates, no adverse clinical signs or detectable abnormalities on haematology and serum biochemistry) for use in water buffalo.     

The adjuvant effect of a single dose of interleukin-12 on murine immune responses to live or killed Brucella abortus strain RB51.

This study was designed to determine if a single 0.5 microg administration of recombinant murine interleukin-12 (IL-12) would influence immune responses of mice vaccinated with live or killed Brucella abortus strain RB51 (SRB51). Mice were vaccinated intraperitoneally with 5 x 10(8) cfu of live or gamma-irradiated SRB51 bacteria alone, or in combination with 0.5 microg of IL-12. Control mice received saline or 0.5 microg of IL-12. Serologic responses and spleen weights after vaccination were greater in mice vaccinated with live SRB51 when compared to mice receiving killed SRB51 or control treatments. Administration of a single dose of IL-12 as a vaccine adjuvant did not influence immune responses, clearance of live SRB51, or resistance against B. abortus strain 2308 (S2308) challenge. The results of this study suggest that a single administration of 0.5 microg of IL-12 at the time of vaccination does not have significant adjuvant effects on vaccine-induced immune responses against live or killed Brucella.     

Brucellosis induced by RB51 vaccine in a pregnant heifer

Brucellosis developed in a 14.5-month-old Gelbvieh heifer after the animal was vaccinated with the calfhood dose of strain RB51 Brucella abortus vaccine s.c. during the fourth month of its first pregnancy. The heifer experienced dystocia and was euthanatized during cesarean section because of a large uterine tear. The fetus was dead at delivery. Suppurative placentitis and fetal pneumonia were evident at necropsy. Brucella abortus strain RB51 was isolated from the placenta and the fetus' lung.     

Use of detergent extracts of Brucella abortus RB51 to detect serologic responses in RB51-vaccinated cattle.

Serologic responses to the newly introduced rough Brucella abortus vaccine strain RB51 have been determined in a dot-blot format using gamma-irradiated RB51 cells as the antigen. Because gamma-irradiated cells are not easily prepared and the signal from cells was not always reliable, an alternative antigen was sought. Detergent extracts of B. abortus RB51 were prepared using zwittergent 3-14, Triton X-100, and sodium dodecyl sulfate (SDS) and examined in a dot-blot format. Zwittergent 3-14 extracts and gamma-irradiated RB51 cells gave the same titers. Unlike gamma-irradiated RB51 cells, zwittergent 3-14 extracts produced signals consistently, and the signals were easily interpreted. Triton X-100 extracts interfered with signal development, and SDS extracts resulted in a high background signal. Western blot analyses revealed several outer membrane proteins in the zwittergent 3-14 extract. The major antigens in the extract had apparent molecular weights of <20,000.     

Parenteral vaccination of domestic pigs with Brucella abortus strain RB51

OBJECTIVE: To determine the immunogenicity and efficacy of Brucella abortus strain RB51 (SRB51) as a vaccine in domestic pigs. ANIMALS: Sixty-eight 6-week-old crossbred domestic pigs and twenty-four 4-month-old gilts. PROCEDURES: In experiment 1, pigs were vaccinated IM (n = 51) with 2 x 10(10) CFUs of SRB51 or sham inoculated (17). Periodic blood samples were obtained to perform blood cultures, serologic evaluations, and cell-mediated immunity assays. Necropsies were performed at selected times between weeks 1 and 23 after vaccination to determine vaccine clearance. In experiment 2, gilts were similarly vaccinated (n = 18) or sham inoculated (8) and similar samples were obtained after vaccination. Gilts were bred and challenged conjunctivally with 5.0 x 10(7) CFUs of virulent Brucella suis strain 3B. Necropsies were performed on gilts and on fetuses or neonates after abortion or parturition, respectively. Bacterial cultures and serologic evaluations were performed on samples obtained at necropsy to determine vaccine efficacy. RESULTS: Humoral and cell-mediated immune responses did not differ between vaccinates and controls. After vaccination, SRB51 was not isolated from blood cultures of either group and was isolated from lymphoid tissues of 3 pigs at 2 weeks (n = 2) and 4 weeks (1) after vaccination. No differences were found in isolation of B suis or in seroconversion between vaccinated and control gilts and between their neonates or aborted fetuses. CONCLUSIONS AND CLINICAL RElEVANCE: Parenteral vaccination with SRB51 does not induce humoral or cell-mediated immune responses. Vaccination with SRB51 did not protect gilts or their neonates and fetuses from virulent challenge with B suis.     

Interaction of bovine chorioallantoic membrane explants with three strains of Brucella abortus.

Chorioallantoic membrane (CAM) explants were used to determine the in vitro growth and cytotoxic potential of 3 strains of Brucella abortus. Bovine CAM explants were inoculated with 2 x 10(7) colony-forming units of the pathogenic strain 2308, attenuated strain 19, or the rough strain RB51 of B abortus. After inoculation, the explants were harvested and examined at 2 or 4 hours, 12 or 14 hours, and 24 or 26 hours of incubation. Bacterial growth associated with each explant was determined by counting colony-forming units. The degree of cellular damage in each explant associated either with bacterial growth or bacterial toxins was evaluated by morphometric analysis after trypan blue staining. Significant differences were not detected in the numbers of bacteria of any strain of B abortus in the CAM explants at comparable time intervals. The rate of growth of the bacteria in CAM explants was higher between 2 and 12 hours after inoculation than between 12 and 24 hours after inoculation. Cytotoxic effects associated with strain 2308 were significantly (P less than 0.05) greater than that caused by other strains. Cytotoxic effects associated with strain 19 and rough strain RB51 were similar, and both were significantly (P less than 0.05) greater than the phosphate buffer solution control. Chorioallantoic membrane explants inoculated with a filtrate of heat-killed strain 2308 induced minimal cellular damage, compared with that caused by the viable bacteria. These results indicated that the number of B abortus in trophoblasts was independent of the degree of cellular damage.     

Serological and bacteriological responses of water buffalo (Bubalus bubalis) vaccinated with two doses of Brucella abortus strain RB51 vaccine

Thirty-two water buffalo (Bubalus bubalis) calves aged 6?C10?months were used to evaluate serological responses to Brucella abortus strain RB51 (RB51) vaccination in a dose?Cresponse study and to compare the use of two selective media for the isolation of RB51. The animals were randomly divided into three treatment groups. Groups I?CIII received the recommended vaccine dose (RD) twice 4?weeks apart, RD twice 18?weeks apart and saline once, respectively. Lymph nodes were excised from the three groups and subjected to bacteriological examination to determine the frequency of detection of RB51. Pre- and post-vaccination blood samples were collected and tested for B. abortus antibodies using the buffered plate agglutination test (BPAT), complement fixation test (CFT), and dot-blot assay. Sera taken at all post-inoculation weeks (PIW) were negative for field strain B. abortus using the BPAT. Antibody responses to RB51 were demonstrated in all vaccinates but not in controls by CFT and dot-blot assay from 1 PIW up to 16?weeks following booster vaccination. The agreement for both assays was 80.7% and there was a linear interdependence with a Pearson??s correlation coefficient value of 0.578. The frequency of isolation of RB51 from the two selective media used was not significantly different (P?>?0.05).     

Responses of cattle to two dosages of Brucella abortus strain RB51: serology, clearance and efficacy

A new brucellosis vaccine, Brucella abortus strain RB51 (SRB51), is currently recommended for use as a calfhood vaccine in the US at dosages between 1 x 10(10)and 3.4 x 10(10)colony-forming units (CFU). The purpose of the study reported here was to compare responses to minimal and maximal recommended SRB51 dosages. Eighteen heifer calves were vaccinated subcutaneously with 1.6 x 10(10)CFU of SRB51, 3.2 x 10(10)CFU of SRB51, or saline (n = 6 per treatment). The vaccine strain was recovered from the superficial cervical lymph node 14 weeks after vaccination in two of six animals that received 1.6 x 10(10)CFU SRB51, but not from any cattle vaccinated with 3.2 x 10(10)CFU SRB51. The higher SRB51 dosage stimulated greater antibody titres. Protection against abortion or infection following B. abortus strain 2308 (S2308) challenge was similar for both SRB51 dosages and greater than resistance of non-vaccinates. The vaccine strain was recovered from one heifer and her fetus at necropsy 1 week prior to estimated parturition. Data from this study suggests that SRB51 induces similar protective immunity across the recommended dosage range. The SRB51 vaccine may persist in some cattle into adulthood but the incidence and significance of this persistence remains unknown.