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.     

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.     

An aerosolized Brucella spp. challenge model for laboratory animals

To characterize the optimal aerosol dosage of Brucella abortus strain 2308 (S2308) and B. melitensis (S16M) in a laboratory animal model of brucellosis, dosages of 10(3)-10(10) colony forming units (CFU) were nebulized to mice. Although tissue weights were minimally influenced, total CFU per tissues increased beginning at 10(6)-10(7) CFU dosages, with 10(9) CFU appearing to be an optimal dosage for S16M or S2308 aerosol delivery. At 12 weeks after vaccination with 10(7) CFU of B. abortus strain RB51 (SRB51) or saline (control), mice were challenged intraperitoneally (i.p.) (6.4 x 10(4) CFU) or via aerosol (1.76 x 10(9) CFU) with S2308. Mice vaccinated with SRB51 had reduced (P < 0.05) splenic, liver and lung colonization (total CFU and CFU/g) after i.p. challenge with S2308 as compared with control mice after i.p. S2308 challenge. Control and SRB51-vaccinated mice did not differ (P > 0.05) in splenic, liver or lung colonization after aerosol S2308 challenge. Failure to demonstrate vaccine protection was not because of a high aerosol challenge dosage as colonization of spleen and liver tissues was lower (P < 0.05) after aerosol challenge when compared with control mice after i.p. S2308 challenge.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Anti-chlamydial immunity in ewes conferred by vaccination with a combination of three live chlamydia, brucella and salmonella vaccines

Live attenuated vaccines against Chlamydia psittaci var ovis, Brucella melitensis and Salmonella abortus ovis have previously been shown to be compatible in mice by subcutaneous administration. Immunity against challenge with virulent chlamydia was, however, slightly decreased in associations including the B melitensis Rev 1 vaccine. The chlamydia strain 1B vaccine was administered to four- to five-month-old female lambs, either alone or in combination with the B melitensis Rev1 and the S abortus ovis Rv6 vaccines. Clinical, serological and bacteriological observations demonstrated the compatibility of the three vaccines. Control, singly and triply vaccinated ewes were challenged with a virulent strain of chlamydia during their second pregnancy, 15 months after vaccination. Five of the 12 control ewes lambed normally and 10 of them were infected, as shown by the excretion of the challenge chlamydia in genital secretions. Sixteen of the 17 ewes in the triple vaccine group lambed normally and none was infected. All 12 in the single vaccine group lambed normally and three of the 12 were infected. In spite of this unusually poor protection by the single vaccine, antichlamydial immunity was clearly not decreased by the association with the two other vaccines.     

Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis

To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.     

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.     

Identification of a virulence factor for Brucella abortus infection in BALB/c mice

Immunogenic or pathogenic factors of recombinant proteins (rBCSP20, rBCSP-31, and rBCSP45 of Brucella abortus strain 19) for mice were compared with factors of a proteinase K-treated lipopolysaccharide extracted from B abortus strain 2308. Mice were vaccinated with 4 products, using different inoculation schedules and were challenge exposed with a virulent culture of B abortus strain 2308. Blood samples were collected 2 weeks after vaccination and at necropsy and sera were obtained. Spleens were cultured for B abortus at necropsy (3 to 4 weeks after challenge exposure). Mice given proteinase K-treated lipopolysaccharide alone or in conjunction with rBCSP20 or rBCSP45 proteins were protected, but mice given rBCSP31 on the same day as challenge exposure were not. Vaccination with recombinant proteins alone neither provide protection nor significantly (P greater than 0.05) increase the pathogenic effect of the challenge-exposure culture. Seemingly, rBCSP31 might be a virulence factor of B abortus.     

Protection of mice against Brucella abortus infection by inoculation with monoclonal antibodies recognizing Brucella O-antigen

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.     

Unresponsiveness of vaccinated BALB/c mice to a second inoculation of lipopolysaccharide from Brucella abortus strain 2308

A study was conducted to determine whether the protection induced in mice by a primary inoculation of lipopolysaccharide from Brucella abortus would be enhanced by a second inoculation given at different time intervals. Protection was challenged by exposure of the mice to a virulent culture of B. abortus strain 2308. Reduced mean viable count and/or splenic weights were the criteria of protection. There was no significant difference (P greater than 0.05) in the protective responses among mice given a single inoculation. Vaccinated mice were significantly (P less than 0.05) better protected than were nonvaccinated mice. Mice given vaccinal inoculations simultaneous with challenge exposure were less protected (P less than 0.001) than were mice vaccinated prior to challenge, but were better protected (P less than 0.010) than were nonvaccinated mice.     

Evaluation of a rough mutant of Brucella melitensis in pregnant goats

Brucella melitensis strain VTRM1, a rough derivative of B melitensis strain 16M, is able to colonise the lymph nodes of goats, does not induce abortion in pregnant goats when used at doses leading to abortions with virulent strain 16M, and does not induce anti-O chain antibodies. However, strain VTRM1 as a single dose vaccine induces only partial protection against both infection and abortion following challenge.     

Protection levels in vaccinated heifers with experimental vaccines Brucella abortus M1-luc and INTA 2

Brucella abortus M1-luc is a mutant strain derived from S19 vaccine strain in which most of bp26 sequence has been replaced by the luciferase coding gene. Strain I2 is a double mutant derived from M1-luc in which most of omp19 has been deleted without introduction of any genetic markers. In BALB/c mice, M1-luc presented equivalent performance to S19 regarding persistence, splenomegaly and protection against challenge. Interestingly, I2 was more attenuated than S19, with no reduction of protection against challenge. In order to evaluate the potential for vaccine use of these strains in the natural host, four groups of 15 heifers, 6-month old, were either non-vaccinated or vaccinated with S19, M1-luc or I2. To at reached 17-month old, heifers were synchronized with two doses of PGF2alpha and received natural service during 60 days with two bulls. Pregnant heifers were challenged at approximately six gestation months with virulent B. abortus S2308. Blood samples post-challenge of heifers were collected for serologic test as well as specimens of aborted fetuses and premature calves for bacterial isolation and histopathological analyses. Protection levels against abortion were 78.6% for S19, 81.8% for M1-luc and 45.5% for I2, compared to the 25% that did not abort from the non-vaccinated group. These results indicate that in bovines BP26 had no influence in protective capacity of S19, correlating with the results obtained in mice. However, contrarily to what was previously observed in mice, lack of expression of Omp19 rendered in less protection capacity of S19 in the natural host.     

Effects of exogenous recombinant interleukin-12 on immune responses and protection against Brucella abortus in a murine model.

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 gamma-irradiated SRB51 bacteria alone, or with IL-12 (0.5 or 1.0 microg, 2x or 3x), whereas other mice received saline or IL-12 alone. Post-vaccination antibody responses to live or killed SRB51 and clearance of live SRB51 from splenic tissue were not influenced by IL-12 treatments. Mice were challenged at 12 weeks with 4 x 10(4) cfu of B. abortus strain 2308 (S2308) and were euthanized 2 weeks later. The highest IL-12 treatment increased (P < 0.05) post-challenge antibody responses when co-administered with killed SRB51. Co-administration of 1.0 microg of IL-12 with live SRB51, but not killed SRB51, reduced (P < 0.05) S2308 colonization of splenic tissues. Our data suggest that although IL-12 may augment protective immunity induced by live SRB51, it does not influence protection induced by vaccination with killed SRB51.     

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.