Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.     

Brucella abortus RB51: enhancing vaccine efficacy and developing multivalent vaccines

Brucella abortus vaccine strain RB51 is an attenuated, stable rough mutant that is being used in many countries to control bovine brucellosis. Our earlier study demonstrated that the protective efficacy of strain RB51 can be significantly enhanced by overexpressing Cu–Zn superoxide dismutase (SOD), a homologous protective antigen. We have also previously demonstrated that strain RB51 can be engineered to express heterologous proteins and mice vaccinated with such recombinant RB51 strains develop a strong Th1 type of immune response to the foreign proteins. The present study is aimed at combining these two characteristics to generate new recombinant RB51 vaccines with enhanced abilities to protect against brucellosis and simultaneously able to protect against infections by Mycobacterium spp. We constructed two recombinant RB51 strains, RB51SOD/85A which overexpresses SOD with simultaneous expression of the 85A, a protective protein of Mycobacterium spp., and RB51ESAT which expresses ESAT-6, another protective protein of M. bovis, as a fusion protein with the signal sequence and few additional amino terminal amino acids of SOD. Mice vaccinated with these recombinant strains developed specific immune responses to the mycobacterial proteins and significantly enhanced protection against Brucella challenge compared to the mice vaccinated with strain RB51 alone.     

Evaluation of brucellosis RB51 vaccine for domestic water buffalo (Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo (Bubalus bubalis) were obtained from a brucellosis-free farm to determine effectiveness of RB51 vaccination for prevention of Brucella infection under natural-exposure conditions in Trinidad. Study animals (20 males and 12 females 5–20 months old) were assigned to vaccination or control groups, using a block randomization design ensuring equal sex distributions between groups. The vaccination group received commercially available RB51 at the recommended calfhood dose of (1.0–3.4)×10¹⁰ colony-forming units (CFU) and controls received 2 ml sterile saline. Vaccination did not result in positive serologic results as measured by four traditional agglutination tests: standard tube agglutination test (STAT), standard plate agglutination test (SPAT), buffered plate agglutination test (BPAT), and card agglutination. Study animals were maintained in a brucellosis-positive herd in southern Trinidad with an estimated 56% prevalence to allow for natural exposure to B. abortus, which was evaluated using STAT, SPAT, BPAT, and card tests. Animals were sampled seven times over 2 years and were classified as positive if they had persistent agglutination titers or had Brucella isolated from specimens collected at completion of the study. Five of the original 32 study animals were lost to follow-up during the field trial. Six of the 14 (43%) vaccinated animals completing the study were classified as positive for Brucella infection—as were two of the 13 (15%) control animals (P=0.21). Isolates from four vaccinates and one control were confirmed as B. abortus biovar 1.     

Efficacy of calfhood vaccination with Brucella abortus strain RB51 in protecting bison against brucellosis

In the studies reported here, protection induced by calfhood vaccination of bison with 1.2-6.1 x 10(10)CFU of Brucella abortus strain RB51 (SRB51) against a virulent strain of B. abortus was evaluated. Non-vaccinated and SRB51-vaccinated bison were intraconjunctivally challenged during midgestation with 3 x 10(7)CFU of virulent B. abortus strain 2308 (S2308). Maternal and fetal tissues were obtained within 24hour after abortion or parturition. Incidence of abortion was greater (P<0.05) in non-vaccinated as compared to SRB51-vaccinated bison (62% and 15%, respectively), with abortions occurring between 5 and 8 weeks after experimental challenge. Calves from bison vaccinated with SRB51 had a reduced (P<0.05) prevalence of fetal infection with S2308 as compared to calves from non-vaccinated bison (19% and 62%, respectively). Although the ability to recover the 2308 challenge strain from maternal tissues did not differ (P>0.05) between nonvaccinates and vaccinates (100% and 78%, respectively), calfhood vaccination with SRB51 reduced (P<0.05) recovery of S2308 from uterine or mammary gland tissues. In bison which did not abort, S2308 was routinely recovered in low numbers from maternal lymphatic tissues; particularly the parotid, bronchial, supramammary, and mandibular lymph nodes. The RB51 vaccine strain was not recovered at any time from maternal or fetal samples obtained at necropsy. Histological lesions associated with Brucella-induced abortions were suppurative placentitis, fetal broncho-interstitial pneumonia, and fetal histiocytic splenitis. The results of our studies suggest that calfhood vaccination of bison with SRB51 is efficacious in protecting against intramammary, intrauterine, and fetal infection following exposure to a virulent strain of B. abortus during pregnancy. As brucellosis is transmitted horizontally through fluids associated with the birth or abortion of an infected fetus, or vertically to the calf through the ingestion of milk containing B. abortus, our data suggest that calfhood vaccination with SRB51 will be beneficial in preventing transmission of brucellosis in bison.     

Efficacy of strain RB51 vaccine in protecting infection and vertical transmission against Brucella abortus in Sprague-Dawley rats

Immunizing animals in the wild against Brucella (B.) abortus is essential to control bovine brucellosis because cattle can get the disease through close contact with infected wildlife. The aim of this experiment was to evaluate the effectiveness of the B. abortus strain RB51 vaccine in protecting infection as well as vertical transmission in Sprague-Dawley (SD) rats against B. abortus biotype 1. Virgin female SD rats (n = 48) two months of age were divided into two groups: one group (n = 24) received RB51 vaccine intraperitoneally with 3 × 10¹⁰ colony forming units (CFU) and the other group (n = 24) was used as non-vaccinated control. Non-vaccinated and RB51-vaccinated rats were challenged with 1.5 × 10⁹ CFU of virulent B. abortus biotype 1 six weeks after vaccination. Three weeks after challenge, all rats were bred. Verification of RB51-vaccine induced protection in SD rats was determined by bacteriological, serological and molecular screening of maternal and fetal tissues at necropsy. The RB51 vaccine elicited 81.25% protection in SD rats against infection with B. abortus biotype 1. Offspring from rats vaccinated with RB51 had a decreased (p < 0.05) prevalence of vertical transmission of B. abortus biotype 1 compared to the offspring from non-vaccinated rats (20.23% and 87.50%, respectively). This is the first report of RB51 vaccination efficacy against the vertical transmission of B. abortus in the SD rat model.     

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.     

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.     

Cu/Zn SOD蛋白在布鲁菌S19疫苗株中的过表达及鉴定

以Cu/Zn SOD为目的基因,通过基因工程技术分别构建了组成型过表达系统pBBR-trc-sod和诱导型过表达系统pBBR-lacPtrc-sod,在布鲁菌S19疫苗株中进行了Cu/Zn SOD的过表达。同时,以大肠杆菌为宿主表达纯化的重组Cu/Zn SOD蛋白免疫家兔制备多克隆抗体,对2种过表达系统中Cu/Zn SOD蛋白的表达量进行分析。结果显示,诱导型Cu/Zn SOD过表达系统的表达量更高,且能与所制备的多抗发生特异性反应。结果表明,构建的过表达系统能够实现Cu/Zn SOD蛋白在布鲁菌S19疫苗株中过表达;同时,该试验也提示我们这种表达系统可应用于布鲁菌S19疫苗的抗原改造。     

Biosafety of Brucella abortus strain RB51 for vaccination of mature bulls and pregnant heifers.

OBJECTIVE: To determine shedding and colonization profiles in mature sexually intact bulls and pregnant heifers after vaccination with a standard calfhood dose of Brucella abortus strain RB51 (SRB51). ANIMALS: 6 sexually mature 3-year-old Jersey bulls and 7 mixed-breed heifers in midgestation. PROCEDURE: Bulls and pregnant heifers were vaccinated IM with the standard calfhood dose of 3x10(10) colony-forming units of SRB51. After vaccination, selected body fluids were monitored weekly for vaccine organism shedding. Pathogenesis was monitored in bulls by weekly breeding soundness examination and, in heifers, by delivery status of the calf. Vaccine organism colonization was assessed by obtaining select tissues at necropsy for bacterial culture. Serologic analysis was performed by use of numerous tests, including complement fixation, an SRB51-based ELISA, and immunoblot analysis. RESULTS: After vaccination, none of the vaccinated bulls or heifers shed SRB51 in their secretions. Results of breeding soundness examination for bulls were normal as was delivery status of the pregnant heifers (6 live births, 1 dystocia). At necropsy, SRB51 was not recovered from any of the selected tissues obtained from bulls, heifers, or calves; however, serologic analysis did detect SRB51-specific antibodies in all cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Vaccination with the standard calfhood dose of SRB51 administered IM was not associated with shedding or colonization in sexually mature bulls or pregnant heifers. Also, under conditions of this study with small numbers of animals, IM vaccination with SRB51 does not appear to cause any reproductive problems when administered to sexually mature cattle.     

Biosafety and antibody responses of adult bison bulls after vaccination with Brucella abortus strain RB51.

OBJECTIVE: To evaluate clearance, antibody responses, potential shedding, and histologic lesions in reproductive tissues of adult bison bulls after vaccination with Brucella abortus strain RB51. ANIMALS: 61 two- and 3-year-old bison bulls. PROCEDURE: 12 bison bulls were vaccinated s.c. with B abortus strain RB51, 3 were inoculated s.c. with 0.15 M NaCl, and antibody responses were evaluated. Various specimens were obtained to evaluate bacterial shedding. Four vaccinates and 1 control were necropsied 10, 20, and 30 weeks after vaccination. In a separate experiment, bison bulls were vaccinated s.c. with 0.15 M NaCl, or by hand or ballistically with strain RB51. Antibody responses were monitored 6 weeks after vaccination and during necropsy 13 weeks after vaccination. Tissue specimens obtained during necropsy from both studies were evaluated bacteriologically and histologically. RESULTS: Strain RB51 was recovered at various times from semen of 3 of 12 vaccinated bison bulls in experiment 1. During necropsy, strain RB51 was recovered 10 and 20, but not 30, weeks after vaccination. In experiment 2, strain RB51 was recovered from lymphoid tissues of hand- and ballistic-vaccinated bison bulls during necropsy. In both experiments, microscopic lesions in testes, epididymis, and seminal vesicles were minimal and did not differ between strain RB51-vaccinated and saline-inoculated bison bulls. CONCLUSIONS AND CLINICAL RELEVANCE: Strain RB51 does not induce relevant inflammatory lesions in reproductive tissues of adult bison bulls. Shedding of strain RB51 in semen may be transient in some bison bulls; however, the importance of this observation is unknown.     

流产布鲁氏菌疫苗候选株RB6生物学特性研究

为了开发布鲁氏菌病新型标记疫苗,本研究对流产布鲁氏菌基因缺失株RB6的培养特性、染色特性、凝集特性、稳定性及小鼠体内毒力和免疫保护力进行了系统鉴定,旨在阐明该菌株具备的生物学特性。通过对亲本菌株和RB6的比较,发现固体培养基上RB6菌株单菌落可被结晶紫染成紫色,RB6菌株液体培养物可与0.1%吖啶黄染料以及抗布鲁氏菌粗糙型抗体发生凝集反应,证明该菌株为粗糙型。将RB6菌株在体外连续传代培养20次和牛体内连续5次继代,检测结果证明其表型未发生变化,说明该菌株遗传稳定性良好。通过小鼠体内试验发现该菌株毒力显著降低,并对流产布鲁氏菌强毒菌株2308攻毒的免疫保护力与现有疫苗A19接近。本研究结果表明,流产布鲁氏菌基因缺失株RB6为粗糙型菌株,毒力较低、安全性高、遗传性状稳定,并具有良好的免疫保护效力,有望开发成为动物布鲁氏菌病粗糙型标记疫苗。     

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.     

The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model

Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.     

Antibodies in calves on feed supplemented with chlortetracycline after vaccination with Brucella abortus strain 19

Twenty dairy heifers each consumed 350 mg of chlortetracycline/day in their feed. Four tests were performed on serum specimens from these and 20 control calves after vaccination with Brucella abortus strain 19. The numbers of positive test results on the card test and mean titers on the tube and rivanol agglutination and complement-fixation tests were compared in the 2 groups. Using the rivanol and complement-fixation tests, there were differences in the mean titers at weeks 5 and 6 after vaccination, but by week 10, differences were not found. The results suggest that addition of low concentration of chlortetracycline in feeds have minimal effects on postvaccinal serologic reactions determined after strain-19 inoculation.     

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.     

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.     

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.