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Ovine brucellosis caused by Brucella ovis is a major cause of reproductive failure in sheep. This study aimed to evaluate transplacental infection and pathogenicity of B.ovis wild type strain ATCC 25,840 (WT B.ovis) and the candidate vaccine strain B.ovis ΔabcBA in pregnant mice. A total of 40 BALB/c mice were equally divided into 4 groups: (i) non immunized and uninfected control mice (3/10 mice became pregnant); (ii) non immunized and challenged with WT B.ovis (5/10 pregnant); (iii) inoculated only with B.ovis ΔabcBA (6/10 pregnant); (iv) immunized with B.ovis ΔabcBA and challenged with WT B.ovis (5/10 pregnant). Female mice bred, and five days after visualization of the vaginal plug, they were inoculated intraperitoneally (ip) with 100 µL of sterile PBS, 100 µL of 1 × 10

Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely, ribosomal protein L7/L12, outer membrane protein (OMP) 22, OMP25 and OMP31, was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. Four proteins were cloned, expressed and purified, and their immunocompetence was analysed. BALB/c mice were immunized subcutaneously with single subunit vaccines (SSVs) or CSV. Cellular and humoral immune responses were determined by ELISA. Results of immunoreactivity showed that these four recombinant proteins reacted with Brucella-positive serum individually but not with Brucella-negative serum. A massive production of IFN-γ and IL-2 but low degree of IL-10 was observed in mice immunized with SSVs or CSV. In addition, the titres of IgG2a were heightened compared with IgG1 in SSV- or CSV-immunized mice, which indicated that SSVs and CSV induced a typical T-helper-1-dominated immune response in vivo. Further investigation of the CSV showed a superior protective effect in mice against brucellosis. The protection level induced by CSV was significantly higher than that induced by SSVs, which was not significantly different compared with a group immunized with RB51. Collectively, these antigens of Brucella could be potential candidates to develop subunit vaccines, and the CSV used in this study could be a potential candidate therapy for the prevention of brucellosis.

The northern areas of China are traditional endemic regions for brucellosis in both animals and humans, while occasional outbreaks of brucellosis have been observed in neglected southern provinces. On 16 December 2020, Chongqing Center for Disease Control and Prevention (CQCDC) received a report of 15

Brucellosis caused by Brucella abortus and Brucella melitensis is endemic in the Republic of Azerbaijan but a complex mix of fiscal, political and technical constraints has impeded regulatory authority decision making for adoption of a sustainable national control programme. This paper reports a series of epidemiologic studies of the disease in animals and humans which we conducted between 2009 and 2020. A preliminary study and a subsequent larger study using vaccination of all non-pregnant female sheep and goats of breeding age and all females between 3 and 8 months with conjunctival Rev1 vaccine both recorded significant reduction in small ruminant seroprevalences. A case control study of winter pasture flocks found many case and control farmers used raw milk to make dairy products for sale, ate fresh cheese and sold dairy products in unregulated markets. Almost all farmers expressed willingness to pay a portion of the costs associated with elimination of brucellosis from their flocks. A pilot human study in 2009 led to a large study in 2017 which recorded an overall seroprevalence of 8.1% in humans. Persons in farm related occupations were at greater risk than urban persons and males were more likely to be seropositive than females. Risk factors included keeping small ruminants, using raw milk cheese and slaughtering animals whereas having heard education information about brucellosis and vaccinating against brucellosis were protective.

Brucellosis is a zoonotic infection caused by the consumption of contaminated raw milk and dairy products. This study aims to compare survival rates of

Brucellosis is a zoonotic disease causes by Brucella bacteria. So far, there is not any efficient treatment for this infectious disease in animals, although subunit vaccines can be a safe alternative. To this aim, we have designed a new chimeric OMP25‐OMP31 antigen formulated in Chitosan nanoparticles and studied its protective efficiency in vivo. OMP25–OMP31 was produced using spliced overlap extension by polymerase chain reaction and the 3D protein structure and antigenic ability were predicted using computational tools. In addition, the humoural and cellular immune responses were measured by ELISA in six different experimental groups. The immune response showed chimeric rOMP25–OMP31 antigen‐induced higher titers of IFN‐γ and TNF‐α cytokines, while the lowest amount of IL‐4 was dedicated to itself. Also, rOMP25–OMP31 stimulated higher titer of IgG than individual injection of rOMP25 and rOMP31 treatments and the cell proliferation assay demonstrated the vaccination with rOMP25–OMP31 elicits a vigorous antigen‐specific cell proliferative. In addition, the challenge experiment showed immunised mic stimulated a higher level of protection than negative controls. Overall, the results of rOMP25–OMP31 could be promising for considering chimeric constructs as a feasible vaccine candidate for further investigations against brucellosis., Chimeric antigen induced better immune response.

Brucella is an intracellular zoonotic pathogen that can affect many hosts. Brucella melitensis Rev.1 is a live attenuated, is one of the most effective vaccine strain against brucellosis. It can be used safely in sheep, goats, and even cattle. Although many studies are available on this topic, there is no effective vaccine strain for sheep and goats that distinguishes the antibody titer produced between the field infections and vaccinations. Outer membrane protein 19 (Omp 19) is both virulent and a protective antigen found on the cell-wall of the Brucella strain. In this study, used the suicide plasmid pJQ200KS, which contained homologous region without Omp19 Open Reading Frame (ORF) that was transferred to B. melitensis Rev.1 and further transformed into spheroplasts along with penicillin, ampicillin, and glycine by electroporation. To obtain a mutant vector from Escherichia coli, we used the heat shock transformation method along with the blue-white colony screening using X-gal media, whereas for the gene transfer in Brucella, we used electroporation. A scanning electron microscope (S.E.M) was used to observe the spheroplast transformation while the mutant vector and deletion mutants were confirmed through PCR and sequence analysis. In the mouse model efficacy trials, three commercial vaccines were found to comply with the OIE standards. Although the deletion mutants 19 and 44/10 had similar efficiency as the commercial vaccines in terms of stimulation power, the ELISA test with Omp19 protein showed the same results as the negative control. The Rev.1 Omp19 deletion mutants obtained in this study contained sufficient residual virulence, and their protective immunity was similar to the commercial vaccines. The study showed that a vaccine prepared using a B. melitensis Rev.1 ΔOmp19 can act as a marker vaccine or differentiate infected from vaccinated animals (DIVA) through the ELISA test that detects the Omp19 protein.

Background A new candidate vector vaccine against human brucellosis based on recombinant influenza viral vectors (rIVV) subtypes H5N1 expressing Brucella outer membrane protein (Omp) 16, L7/L12, Omp19 or Cu–Zn SOD proteins has been developed. This paper presents the results of the study of protection of the vaccine using on guinea pigs, including various options of administering, dose and frequency. Provided data of the novel vaccine candidate will contribute to its further movement into the preclinical stage study. Methods General states of guinea pigs was assessed based on behavior and dynamics of a guinea pig weight-gain test. The effectiveness of the new anti-brucellosis vector vaccine was determined by studying its protective effect after conjunctival, intranasal and sublingual administration in doses 105 EID50, 106 EID50 and 107 EID50 during prime and boost vaccinations of animals, followed by challenge with a virulent strain of B. melitensis 16 M infection. For sake of comparison, the commercial B. melitensis Rev.1 vaccine was used as a control. The protective properties of vaccines were assessed by quantitation of Brucella colonization in organs and tissues of infected animals and compared to the control groups. Results It was observed a gradual increase in body weight of guinea pigs after prime and booster immunization with the vaccine using conjunctival, intranasal and sublingual routes of administration, as well as after using various doses of vaccine. The most optimal way of using the vaccine has been established: double intranasal immunization of guinea pigs at a dose of 106 EID50, which provides 80% protection of guinea pigs from B. melitensis 16 M infection (P B. melitensis Rev.1 vaccine. Conclusions We developed effective human vaccine candidate against brucellosis and developed its immunization protocol in guinea pig model. We believe that because of these studies, the proposed vaccine has achieved the best level of protection, which in turn provides a basis for its further promotion.

Purpose Brucellosis as a worldwide zoonotic illness affect domestic animals and humans doesn't have any vaccine for the prevention of infection in humans yet. The aim of this study was to evaluate the specific immune response following the administration of glycine nanoparticles as adjuvant and delivery system of a chimeric antigen contained trigger factor, Omp31, and Bp26 in murine model. Materials and methods The chimeric antigen of Brucella was cloned and expressed in Escherichia coli (E. coli) BL21 (DE3). Purification and characterization of recombinant protein was conducted through Ni-NTA (nickel-nitrilotriacetic acid) agarose, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and Western blot. Nanoparticle characteristics including morphology, particle size distribution, zeta potential, protein retention rate, and release rate were measured in vitro. Subsequently, nanoparticle contained antigen was administered to mice and blood sample was taken to measured the antibody level. Results The protein retention in the nanoparticles was successfully done and the nanoparticle characteristics were appropriate. The average size of glycine particles containing antigen was about 174 nm, and the absorption of protein was approximately 61.27% of the initial value, with a release rate of approximately 70% after 8 hours. Enzyme-linked immunosorbent assay result proved that the immunized sera of mice which were administered with nano-formula contains high levels of antibodies (immunoglobulin G) against recombinant chimeric antigen and also a high level of mucosal antibody (immunoglobulin A) in the oral group, which showed a desirable immunity against Brucella. Conclusion The results showed that chimeric antigen-loaded glycine nanoparticles can act as a vaccine candidate for inducing the cellular and humoral immune response against brucellosis.

Small ruminant brucellosis is caused by the Gram negative cocci-bacillus Brucella (B.) melitensis, the most virulent Brucella species for humans. In goats and sheep, middle to late-term gestation abortion, stillbirths and the delivery of weak infected offspring are the characteristic clinical signs of the disease. Vaccination with the currently available Rev. 1 vaccine is the best option to prevent and control the disease, although it is far from ideal. In this study, we investigate the safety of the B. melitensis 16MΔvjbR strain during a 15-month period beginning at vaccination of young goats, impregnation, delivery and lactation. Forty, 4 to 6 months old, healthy female crossbreed goats were randomly divided into four groups (n = 10) and immunized subcutaneously with a single vaccine dose containing 1x109 CFU of B. melitensis 16MΔvjbR delivered in alginate microcapsules or non-encapsulated. Controls received empty capsules or the commercially available Rev.1 vaccine. Seven months post-vaccination, when animals were sexually mature, all goats were naturally bred using brucellosis-free males, and allowed to carry pregnancies to term. Blood samples to assess the humoral immune response were collected throughout the study. At two months post-delivery, all dams and their offspring were euthanized and a necropsy was performed to collect samples for bacteriology and histology. Interestingly, none of the animals that received the vaccine candidate regardless of the formulation exhibited any clinical signs associated with vaccination nor shed the vaccine strain through saliva, vagina or the milk. Gross and histopathologic changes in all nannies and offspring were unremarkable with no evidence of tissue colonization or vertical transmission to fetuses. Altogether, these data demonstrate that vaccination with the mutant strain 16MΔvjbR is safe for use in the non-pregnant primary host.

BackgroundBrucella spp. is an important zoonotic pathogen responsible for brucellosis in humans and animals.Brucella abortusA19 strain is a widespread vaccine in China. However, it has a drawback of residual virulence in animals and humans.MethodsIn this study, the BALB/c mice were inoculated with either 100 μL PBS(control group, C group), 109 CFU/mL inactivatedB. abortusA19 strain (I group), 105 CFU/mL (low-dose group, L group) 106 CFU/mL liveB. abortusA19 strain (high-dose group, H group), or 105 CFU/mL liveB. abortusA19 strain combined with 109 CFU/mL inactivatedB. abortusA19 strain (LI group). Mice were challenged withB. abortusstrain 2308 at 7 week post vaccination. Subsequently, the immune and protective efficacy of the vaccines were evaluated by measuring splenic bacterial burden, spleen weight, serum IgG, interferon-gamma (IFN-γ), interleukin-4 (IL-4) percentage of CD4 + and CD8 + T cells of mice via bacterial isolation, weighing, ELISA and flow cytometry, respectively.ResultsThe splenic bacterial burden and spleen weight of the mice in group LI were mostly equivalent to the mice of group H. Moreover,Brucella-specific serum IgG, IFN-γ, IL-4, and the percentage of CD4+and CD8+T cells of the LI group mice were similar to those of the H group. In the subsequent challenge test, both vaccines conferred protective immunity to wild-type (WT) 2308 strain. In addition, the levels of IL-4 and IFN-γ, CD4+and CD8+T cells in these mice were similar to those of the mice in the H group.ConclusionsCombined immunization with low dose live vaccine and inactivated vaccine allowed to reduce the liveB. abortusA19 vaccine, dose with an equivalent protection of the high-dose live vaccine.

Fifty-two, Hereford heifers were obtained from brucellosis-free herds and randomly assigned to Brucella abortus strain RB51 (RB51) vaccination (n = 32) or control (n = 20) treatments. Vaccinates received 10

We present the case of an 18-year-old female with a 1-month history of fever, headache, and double vision, whose examination revealed papilledema and cranial nerve VI palsy. Blood cultures grew Brucella abortus cattle vaccine strain RB51, which is inherently resistant to rifampin. We discuss the management of the first known case of neurobrucellosis by this strain.

We evaluated serologic responses of cattle, bison, elk, and swine representing negative control, early vaccination (4–8 wk), late vaccination (21–28 wk) or booster vaccination, early after-experimental challenge (2–4 wk), and late after-experimental challenge (8–21 wk), in a brucellosis fluorescence polarization assay (FPA; n = 10 sera per species per treatment) using negative control sera from cattle, bison, elk, and swine ( n = 5 per species). Sera from cattle shedding Brucella abortus strain RB51 in milk were also evaluated against the 20 negative control sera. The species of negative control sera used in the FPA could increase ( p < 0.05) delta millipolarization (mP; delta mP = sample mP − negative control mP) results. In general, the species of negative control sera did not alter the interpretation of FPA results in control, vaccinated, or infected animals. Even after repeated RB51 vaccinations in bison, cattle, or elk, or in cattle shedding RB51 in milk, serologic results from the FPA remained negative. Species differences in FPA results were noted; elk developed robust humoral responses very quickly after infection that resulted in strong positive FPA results. In cattle and bison, humoral responses appeared to develop over a longer period of time, and greater delta mP values were detected at later times after infection. Sensitivity of the FPA for detecting infected animals was greatest for elk in early challenge samples and bison in late challenge samples. Our data suggest that species of origin of negative control sera does not influence interpretation of the FPA in natural hosts of Brucella abortus.

Small ruminant brucellosis remains endemic in many low- and middle-income countries (LMICs), where it poses a major economic and public health burden. Lack of resources to support long-term vaccination, inherent characteristics of small ruminant production systems such as mixing of different flocks for grazing and limitations of the vaccines currently available, which can induce abortion in pregnant animals, have all hindered the effectiveness of control programmes. In the current study, the likely effect of different control scenarios on the seroprevalence of brucellosis among the small ruminant population in a hypothetical area of an endemic region was simulated using compartmental models. The model accounts for variability in transmission rates between villages and also simulates control scenarios that target villages with high seroprevalence. Our results show that vaccination of young replacement animals only can effectively reduce the prevalence of small ruminant brucellosis in endemic settings if a high vaccination coverage is achieved. On the other hand, test-and-slaughter alone is not a promising strategy for control of small ruminant brucellosis under husbandry practices typical of endemic low-resource settings. Furthermore, results show the potential success of some strategies requiring a relatively low overall vaccination coverage such as the vaccination of 50% of young replacements and 25% of adult animals each year. Control strategies selectively targeting high initial seroprevalence villages (p > 10%) did not decrease the overall seroprevalence to acceptable levels in most of the examined scenarios. Scenario analysis showed that the efficacy of the simulated control strategies can be improved mostly by decreasing the proportion of between-village trade and also by improving the performance of the used serological tests and increasing vaccine efficacy.

Brucellosis is an important occupational disease, mainly among veterinarians, because of their frequent contact with sick animals, contaminated secretions and live attenuated anti-Brucella vaccines. This study aimed to determine the prevalence of accidental exposure to S19 and RB51 vaccine strains and occupational brucellosis among veterinarians registered to administer vaccinations in Minas Gerais, Brazil, as well as to identify the risk factors associated with accidental exposure to anti-Brucella abortus vaccines. Data were collected through an online questionnaire. Three hundred and twenty-nine veterinarians were included in the analyses using stratified random sampling. A multivariate logistic regression analysis was used to evaluate the predictors of accidental exposure to S19 and RB51 strains. Nearly one third of the veterinarians registered to administer bovine brucellosis vaccination in Minas Gerais, 32.83% (108/329) (95% confidence interval [CI]: 27.78-38.19%), reported having been accidentally exposed to S19 or RB51 vaccine strains. The exposure factors associated with this outcome included a score of personnel protective equipment (PPE) use during work (odds ratio [OR], 0.94; 95% CI: 0.89-0.98) and a score of knowledge about brucellosis symptoms, classified as poor (base category), intermediate (OR, 0.26; 95% CI: 0.07-0.87) or good (OR, 0.22; 95% CI: 0.07-0.62). In addition, 4.56% (15/329) (95% CI: 2.57-7.41%) of veterinarians reported that they had brucellosis, of which 46.67% (7/15) considered that the disease was due to accidental exposure to anti-B. abortus live attenuated vaccine. The prevalence of accidental exposure to B. abortus vaccine strains among veterinarians from Minas Gerais enrolled in the control of bovine brucellosis was high. The reduced knowledge about human brucellosis symptoms and lack of appropriate PPE use were risk factors from unintentional contact with S19 and RB51 vaccine strains.

Brucellosis, which is caused by Brucella spp., is an important zoonotic infectious disease that can cause great hazard to public health and safety. However, the current vaccines have several drawbacks, including residual virulence for animals and humans. Bacterial ghost is the empty envelopes of bacteria, which emerge as a proper vaccine candidate. With the purpose of generating B. abortus ghosts and investigating the immunogenicity of bacterial ghosts as vaccine candidate, we used homologous recombination and bacterial ghost technologies to construct 2308ΔgntR ghost strain. Mice were injected with 2308ΔgntR ghost and the safety and immunogenicity of ghost were further evaluated. The mice inoculated with ghost showed no splenomegaly. The 2308ΔgntR ghost induced high protective immunity in BALB/c mice against challenge with S2308, and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of interferon gamma (IFN-γ) and interleukin-4 (IL-4). Additionally, 2308ΔgntR ghost demonstrated strong spleen CD4+ and CD8+ T cell responses. These results suggest that 2308ΔgntR ghost is a potential vaccine candidate and may represent a promising new approach for vaccination against Brucella infection.

Since Brucella infection mostly occurs through the mucosal surfaces, immune response induced by vaccine that is delivered by a way of mucosal route can be drastically enhanced to control the brucellosis. Omp31is the major outer membrane protein of Brucella, and is considered as a protective antigen against Brucella infection. Accordingly, Lactococcus lactis has been used as an antigen-delivering vector to develop a vaccine-induced mucosal response for having a safer vaccination against brucellosis. A designed omp31 gene fused to the usp45 signal peptide and M6 cell wall anchor was sub cloned in the pNZ7021 expression vector, and a recombinant L. lactis displaying Omp31 was constructed. Omp31 protein expression was confirmed using Western blotting and immunofluorescence analysis. Animals were orally and intraperitoneally immunized with live or killed L. lactis expressing Omp31, respectively. The humoral and cellular immune responses were evaluated by measuring the specific cytokines and antibodies. sIgA, serum IgA, IgM, and total IgG antibodies significantly increased in the mice immunized with live recombinant L. lactis expressing Omp31 and also serum IgM, and total IgG antibodies significantly increased in mice immunized with killed recombinant L. lactis expressing Omp31. Among IgG subtypes, IgG2a response was significantly higher in both groups compared to IgG1. In mice groups immunized with recombinant L. lactis, the IFN-γ and IL-10 level elevated; however, there was no change in the level of IL-4. These results indicated that recombinants L. lactis induce both humoral and cellular immune responses in mice, and also vaccines based on L. lactis-derived live carriers are promising interventions against Brucella melitensis infections.

Aim: The aim of the study was to obtain a vaccine against animal brucellosis having high immunogenic properties by carrying an evaluation of the effectiveness of split-conjugated animal brucellosis vaccine combined with fosprenil and polypeptide C as a molecular immunomodulatory adjuvant according to the results of serological studies of the blood of animals: Agglutination reaction, complement fixation, and rose Bengal sample. Materials and Methods: Eighteen calves of Holstein Friesians breed, aged 5 months, with a living weight of 100-150 kg, were divided into three groups of six animals each. All animals were healthy and they received a prophylactic vaccination against brucellosis. The dry split-conjugated vaccine against brucellosis in animals was dissolved in saline and for this purpose, 10 ml of saline was poured into the vaccine vial. Then the content was mixed, and afterward 1 ml was used per animal. Fosprenil was used at the rate of 1 kg of animal weight: 100 kg (calf weight) was multiplied by 0.05 (dose/1 kg of animal weight); 5 ml of fosprenil was obtained, which was collected into disposable syringes and intramuscularly sterilely injected into the croup area. Calves in the first group (control) were intramuscularly injected with the vaccine at a dose of 1.0 ml, and fosprenil at a dose of 5.0 ml was administered intramuscularly once to the croup area. Animals from the second group were subcutaneously immunized by the vaccine with polypeptide C at a dose of 1.0 ml. Polypeptide C is a solution that was poured into a vial with a vaccine at a dose of 10.0 ml, the content was mixed, and then calves were injected subcutaneously into the middle third of the neck in 1 ml (10 doses in a vial). Immunization of calves in the third group was carried out with a vaccine, diluted with an isotonic sodium chloride solution of 0.9%, at a dose of 1.0 ml subcutaneously once. At the 14th, 30th, and 90th days after vaccination, a blood sampling was taken for serological tests: Agglutination test, complement fixation test, and rose Bengal test. Results: After conducting serological studies, it was noted that split-conjugated vaccine against animal brucellosis using fosprenil forms antibodies in large titers and they persist for a longer time in the body of animals compared to the other tested vaccine: The first combination with the immunomodulatory polypeptide C and the vaccine only on the physiological solution. Conclusion: The developed complex of split-conjugated vaccine against brucellosis in animals enhances the humoral immune response of the organism against brucellosis and improves the protection of animals against the disease when it is used with the immunomodulatory fosprenil. In the future, we want to expand the use of the resulting complex in the fight against brucellosis on a larger population and to study the change in cellular immunity after the introduction of the resulting complex on an animal organism.

Brucellosis is an important zoonotic disease that causes great economic losses. Vaccine immunisation is the main strategy for the prevention and control of brucellosis. Although live attenuated vaccines play important roles in the prevention of this disease, they also have several limitations, such as residual virulence and difficulty in the differentiation of immunisation and infection. We developed and evaluated a new bacterial ghost vaccine of

Brucella melitensis and Brucella ovis are gram-negative pathogens of sheep that cause severe economic losses and, although B. ovis is non-zoonotic, B. melitensis is the main cause of human brucellosis. B. melitensis carries a smooth (S) lipopolysaccharide (LPS) with an N-formyl-perosamine O-polysaccharide (O-PS) that is absent in the rough LPS of B. ovis. Their control and eradication require vaccination, but B. melitensis Rev 1, the only vaccine available, triggers anti-O-PS antibodies that interfere in the S-brucellae serodiagnosis. Since eradication and serological surveillance of the zoonotic species are priorities, Rev 1 is banned once B. melitensis is eradicated or where it never existed, hampering B. ovis control and eradication. To develop a B. ovis specific vaccine, we investigated three Brucella live vaccine candidates lacking N-formyl-perosamine O-PS: Bov::CAΔwadB (CO2-independent B. ovis with truncated LPS core oligosaccharide); Rev1::wbdRΔwbkC (carrying N-acetylated O-PS); and H38ΔwbkF (B. melitensis rough mutant with intact LPS core). After confirming their attenuation and protection against B. ovis in mice, were tested in rams for efficacy. H38ΔwbkF yielded similar protection to Rev 1 against B. ovis but Bov::CAΔwadB and Rev1::wbdRΔwbkC conferred no or poor protection, respectively. All H38ΔwbkF vaccinated rams developed a protracted antibody response in ELISA and immunoprecipitation B. ovis diagnostic tests. In contrast, all remained negative in Rose Bengal and complement fixation tests used routinely for B. melitensis diagnosis, though some became positive in S-LPS ELISA owing to LPS core epitope reactivity. Thus, H38ΔwbkF is an interesting candidate for the immunoprophylaxis of B. ovis in B. melitensis-free areas.

Brucellaspp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and animals. Currently available live attenuated vaccines against brucellosis still have drawbacks. Therefore, subunit vaccines, produced using epitope-based antigens, have the advantage of being safe, cost-effective and efficacious. Here, we identifiedB. abortussmall RNAs expressed during early infection with bone marrow-derived macrophages (BMDMs) and an apolipoprotein N-acyltransferase (Int) was identified as the putative target of the greatest expressed small RNA. Decreased expression of Int was observed during BMDM infection and the protein sequence was evaluated to rationally select a putative immunogenic epitope by immunoinformatic, which was explored as a vaccinal candidate. C57BL/6 mice were immunized and challenged withB. abortus, showing lower recovery in the number of viable bacteria in the liver, spleen, and axillary lymph node and greater production of IgG and fractions when compared to non-vaccinated mice. The vaccinated and infected mice showed the increased expression ofTNF-α,IFN-γ, and IL-6 following expression of the anti-inflammatory genesIL-10andTGF-βin the liver, justifying the reduction in the number and size of the observed granulomas. BMDMs stimulated with splenocyte supernatants from vaccinated and infected mice increase the CD86+ marker, as well as expressing greater amounts of iNOS and the consequent increase in NO production, suggesting an increase in the phagocytic and microbicidal capacity of these cells to eliminate the bacteria.

Reverse vaccinology (RV) is the state-of-the-art vaccine development strategy that starts with predicting vaccine antigens by bioinformatics analysis of the whole genome of a pathogen of interest. Vaxign is the first web-based RV vaccine prediction method based on calculating and filtering different criteria of proteins. Vaxign-ML is a new Vaxign machine learning (ML) method that predicts vaccine antigens based on extreme gradient boosting with the advance of new technologies and cumulation of protective antigen data. Using a benchmark dataset, Vaxign-ML showed superior performance in comparison to existing open-source RV tools. Vaxign-ML is also implemented within the web-based Vaxign platform to support easy and intuitive access. Vaxign-ML is also available as a command-based software package for more advanced and customizable vaccine antigen prediction. Both Vaxign and Vaxign-ML have been applied to predict SARS-CoV-2 (cause of COVID-19) and Brucella vaccine antigens to demonstrate the integrative approach to analyze and select vaccine candidates using the Vaxign platform.

Reverse vaccinology (RV) is the state-of-the-art vaccine development strategy that starts with predicting vaccine antigens by bioinformatics analysis of the whole genome of a pathogen of interest. Vaxign is the first web-based RV vaccine prediction method based on calculating and filtering different criteria of proteins. Vaxign-ML is a new Vaxign machine learning (ML) method that predicts vaccine antigens based on extreme gradient boosting with the advance of new technologies and cumulation of protective antigen data. Using a benchmark dataset, Vaxign-ML showed superior performance in comparison to existing open-source RV tools. Vaxign-ML is also implemented within the web-based Vaxign platform to support easy and intuitive access. Vaxign-ML is also available as a command-based software package for more advanced and customizable vaccine antigen prediction. Both Vaxign and Vaxign-ML have been applied to predict SARS-CoV-2 (cause of COVID-19) and Brucella vaccine antigens to demonstrate the integrative approach to analyze and select vaccine candidates using the Vaxign platform.

Vaccination can prevent and control animal brucellosis. Currently, live attenuated vaccines are extensively used to prevent Brucella infection. However, traditional vaccines such as live attenuated vaccines are associated with biological safety risks for both humans and animals. The bacterial ghost (BG) is a new form of vaccine with great prospects. However, bacterial cells cannot be completely inactivated by biological lysis, conferring a safety risk associated with the vaccine. In this study, we developed a Brucella abortus A19 bacterial ghost (A19BG) through a double inactivation strategy with sequential biological lysis and hydrogen peroxide treatment. This strategy resulted in 100% inactivation of Brucella, such that viable bacterial cells were not detected even at an ultrahigh concentration of 10

This is the pre-peer reviewed version of the following article: de Oliveira, M.M., Pereira, C.R., de Oliveira, I.R.C., Godfroid, J., Lage, A.P. & Dorneles, E.M.S. (2021). Efficacy of Brucella abortus S19 and RB51 vaccine strains: A systematic review and meta-analysis. Transboundary and Emerging Diseases, which has been published in final form at https://doi.org/10.1111/tbed.14259. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This systematic review and meta-analysis aimed to recalculate the efficacy of Brucella abortus S19 and RB51 vaccine strains and discuss the main variables associated with controlled trials to evaluate bovine brucellosis vaccine efficacy (VE). The most commonly used vaccine strain was S19, at a dose of 1010 colony forming units (CFU), followed by RB51 at 1010 CFU. The most commonly used challenge strain was B. abortus 2308, at a dose of 107 CFU, by the intraconjunctival route. Regarding the meta-analysis, trials were grouped according to the vaccine strain and dose to recalculate protection against abortion (four groups) or infection (five groups) using pooled risk ratio (RR) and VE. Regarding protection against abortion (n = 15 trials), the S19 vaccine at 109 CFU exhibited the highest protection rate (RR = 0.25, 95% confidence interval (CI) : 0.12–0.52; VE = 75.09%, 95% CI: 48.08–88.05), followed by RB51 at 1010 CFU (RR = 0.31, 95% CI: 0.16–0.61; VE = 69.25%, 95% CI: 39.48–84.38). Regarding protection against infection (n = 23 trials), only two subgroups exhibited significant protection: S19 at 109 CFU (RR = 0.28, 95% CI: 0.14–0.55; VE = 72.03%, 95% CI: 57.70– 81.50) and RB51 at 1010 CFU (RR = 0.43, 95% CI: 0.22–0.84; VE = 57.05%, 95% CI: 30.90–73.30). In conclusion, our results suggest that a dose of 109 CFU for S19 and 1010 CFU for RB51 are the most suitable for the prevention of abortion and infection caused by B. abortus.

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and various animals. The threat of brucellosis has increased, yet currently available live attenuated vaccines still have drawbacks. Therefore, subunit vaccines, produced using protein antigens and having the advantage of being safe, cost-effective and efficacious, are urgently needed. In this study, we used core proteome analysis and a compositive RV methodology to screen potential broad-spectrum antigens against 213 pathogenic strains of Brucella spp. with worldwide geographic distribution. Candidate proteins were scored according to six biological features: subcellular localization, antigen similarity, antigenicity, mature epitope density, virulence, and adhesion probability. In the RV analysis, a total 32 candidate antigens were picked out. Of these, three proteins were selected for assessment of immunogenicity and preliminary protection in a mouse model: outer membrane protein Omp19 (used as a positive control), type IV secretion system (T4SS) protein VirB8, and type I secretion system (T1SS) protein HlyD. These three antigens with a high degree of conservation could induce specific humoral and cellular immune responses. Omp19, VirB8 and HlyD could substantially reduce the organ bacterial load of B. abortus S19 in mice and provide varying degrees of protection. In this study, we demonstrated the effectiveness of this unique strategy for the screening of potential broad-spectrum antigens against Brucella. Further evaluation is needed to identify the levels of protection conferred by the vaccine antigens against wild-type pathogenic Brucella species challenge.

Brucellosis is a worldwide bacterial zoonosis caused by Brucella spp. No approved vaccine is available for human use against the disease. In this study, outer membrane vesicles (OMVs) from a Brucella melitensis biovar 1 human isolate obtained in Iran were used to immunize BALB/c mice (n = 12) by 2 intramuscular injections with a 2-week interval. Another group of 12 mice was used as non-vaccinated controls. Two weeks after the last vaccination, six mice of each group were sacrificed, and proliferation and interferon gamma (IFNγ) production responses of their splenocytes were evaluated following in vitro stimulation with killed Brucella cells. The other mice were challenged with the virulent B. melitensis isolate. Two weeks later, mice were killed and spleens were cultured to determine the number of the challenge strain. The results showed proliferative response and IFNγ production of splenocytes from vaccinated mice (stimulation index: 2.18 ± 0.57, and 1519.35 ± 10.70 pg/mL, respectively) were significantly higher than those of control mice (stimulation index: 1.02 ± 0.02, and 210.01 ± 17.58 pg/mL, respectively). Numbers of the challenge strain in spleens of vaccinated mice were also significantly less than those in the controls with 1.6 units of protection. Our study revealed vaccination with OMVs of the B. melitensis isolate could induce specific immune responses and protection against infection in the mouse model suggesting their potential application for active immunization against brucellosis.

Brucella is an intracellular pathogen that causes abortion in domestic animals and undulant fever in humans. Due to the lack of a human vaccine against brucellosis, animal vaccines play an important role in the management of animal and human brucellosis for decades. Strain 19, RB51 and Rev1 are the approved Brucella spp. vaccine strains that are most commonly used to protect livestock against infection and abortion. However, due to some disadvantages of these vaccines, numerous studies have been conducted for the development of effective vaccines that could also be used in other susceptible animals. In this review, we compare different aspects of immunogenic antigens that have been a candidate for the brucellosis vaccine.

Brucellosis is a worldwide zoonosis affecting animal and human health. Till now, there is no effective vaccine licensed for brucellosis in humans. Although M5, H38 and 45/20 vaccines were used to prevent animal brucellosis in the early stages, the currently used animal vaccines are S19, Rev.1, S2, RB51 and SR82. However, these vaccines still have several drawbacks such as residual virulence and interfering conventional serological tests. With the development of DNA recombination technologies and the completion of the sequence of Brucella genome, much research focuses on the search for potential safer and more effective vaccines. Preliminary studies have demonstrated that new vaccines, including genetically engineered attenuated vaccines, subunit vaccines and other potential vaccines, have higher levels of protection, but there are still some problems. In this paper, we briefly review the main vaccines that have been used in controlling the brucellosis for decades and the progress in the development of new brucellosis vaccines.

Subcellular vaccines against ovine contagious epididymitis due Brucella ovis can solve some shortcomings associated with the use of Brucella melitensis Rev 1. We have demonstrated that the parenteral immunization with polymeric antigen BLSOmp31 emulsified in oil adjuvant conferred significant protection against B. ovis in rams. In our previous studies, we have characterized chitosan microspheres (ChMs) and a thermoresponsive and mucoadhesive in situ gel (Poloxamer 407-Ch) as two novel formulation strategies for the delivery of BLSOmp31 in nasal as well as conjunctival mucosa. In the present work, we evaluated the immunogenicity and protection conferred by the intranasal and conjunctival immunization with these two mucosal delivery systems against B. ovis in rams. BLSOmp31-ChM administered by intranasal route and BLSOmp31-P407-Ch applied by intranasal or conjunctival routes induced systemic, local and preputial IgG and IgA antibody response. Neither formulation showed interference in the serological diagnosis. Thus, mucosal immunization using either formulation induced significant specific cellular immune responses (in vitro and in vivo) and it prevented the excretion of B. ovis in semen. Although these vaccines did not prevent infection in immunized rams, colonization reduction of infected organs and bacterial distribution differed significantly between vaccinated and unvaccinated rams. Fil: Díaz, Alejandra Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva; Argentina Fil: Quinteros, Daniela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Paolicchi, Fernando Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; Argentina Fil: Rivero, Mariana Alejandra. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Palma, Santiago Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Pardo, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Inmunova S.A; Argentina Fil: Clausse, María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Zylberman, Vanesa. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Goldbaum, Fernando Alberto. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Estein, Silvia Marcela. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina

Feral swine cost around $1.5 billion each year in agricultural, environmental, and personal property damages. They are also the most widespread carriers of the zoonotic disease brucellosis, which threatens both livestock bio-security and public health. Currently, there is no approved vaccine against brucellosis in pigs. This is a preliminary report on the development of a live-attenuated B. suis vaccine that could be employed to deliver heterologous antigens to control swine populations. An attenuated vaccine strain provided significant protection against B. suis challenge in mice. Leucine auxotrophy in the vaccine strain allowed the over-expression of heterologous antigens without the use of antibiotic resistant markers. Vaccinated mice showed the development of antibodies against expressed antigen. Further evaluation is required to assess its ability to cause infertility using the mouse model prior to further testing for use as a tool for feral swine population and disease control.

Residual virulence is a major drawback in current Brucella vaccines. Live vaccines induce abortions in pregnant animals. Hence, a novel anti-Brucella vaccine was developed utilizing rough Salmonella delivering four Brucella antigens. Safety implications during pregnancy, humoral immune responses, and protective efficacy against wild type Brucella was investigated in guinea pig model. The vaccine did not induce abortions or severe complications in pregnant guinea pigs when administered 4 × 108 CFU via intraperitoneal route. Systemic IgG determination against antigen components reveals induction of immunity via the Salmonella delivery. Protection efficacy against abortions was 33.3% (2/6) when midterm sow challenged with virulent Brucella 544 strain while none was protected in control group. Lower Brucella recovery in spleen and liver and reduced histopathological burden were also noticed. Although abortion induced by Brucella challenge was not completely prevented, the vaccine candidate may perform better with optimization of vaccination such as inoculation dose optimization.

The live attenuated Brucella melitensis Rev.1 Elberg-originated vaccine strain has been widely used to control brucellosis in small ruminants. However, despite extensive research, the molecular mechanisms underlying the attenuation of this strain are still unknown. In the current study, we conducted a comprehensive comparative analysis of the whole-genome sequence of Rev.1 against that of the virulent reference strain, B. melitensis 16M. This analysis revealed five regions of insertion and three regions of deletion within the Rev.1 genome, among which, one large region of insertion, comprising 3,951 bp, was detected in the Rev.1 genome. In addition, we found several missense mutations within important virulence-related genes, which may be used to determine the mechanism underlying virulence attenuation. Collectively, our findings provide new insights into the Brucella virulence mechanisms and, therefore, may serve as a basis for the rational design of new Brucella vaccines.

Brucellosis is one of the most serious and widespread zoonotic diseases, which seriously threatens human health and the national economy. This study was based on the T/B dominant epitopes of Brucella outer membrane protein 22 (Omp22), outer membrane protein 19 (Omp19) and outer membrane protein 28 (Omp28), with bioinformatics methods to design a safe and effective multi-epitope vaccine. The amino acid sequences of the proteins were found in the National Center for Biotechnology Information (NCBI) database, and the signal peptides were predicted by the SignaIP-5.0 server. The surface accessibility and hydrophilic regions of proteins were analysed with the ProtScale software and the tertiary structure model of the proteins predicted by I-TASSER software and labelled with the UCSF Chimera software. The software COBEpro, SVMTriP and BepiPred were used to predict B cell epitopes of the proteins. SYFPEITHI, RANKpep and IEDB were employed to predict T cell epitopes of the proteins. The T/B dominant epitopes of three proteins were combined with HEYGAALEREAG and GGGS linkers, and carriers sequences linked to the N- and C-terminus of the vaccine construct with the help of EAAAK linkers. Finally, the tertiary structure and physical and chemical properties of the multi-epitope vaccine construct were analysed. The allergenicity, antigenicity and solubility of the multi-epitope vaccine construct were 7.37–11.30, 0.788 and 0.866, respectively. The Ramachandran diagram of the mock vaccine construct showed 96.0% residues within the favoured and allowed range. Collectively, our results showed that this multi-epitope vaccine construct has a high-quality structure and suitable characteristics, which may provide a theoretical basis for future laboratory experiments.

During late July to August 2019, Zhongmu Lanzhou biopharmaceutical plant of Gansu province was producing brucella vaccine for animals and reportedly used an expired disinfectant for cleaning, which failed to kill all the bacteria. At the same time a leak occurred in the plant, which allowed the contaminated waste in the form of aerosol to be leaked in the air. This caused the first outbreak in the Lanzhou animal research institute as the contaminated aerosols were carried and spread there. Community transmission took place after this event. The infection has so far been confirmed in 10,528 people after testing - 68571 people up to November 30, 2020.

Past studies with the live, double-mutant B. abortus (znBAZ) strain resulted in nearly complete protection of mice against pulmonary challenge with wild-type (wt) Brucella via a dominant CD8+ T cell response. To understand the contribution innate immune cells in priming CD8+ T cell responses, mice were nasally dosed with wt B. abortus, smooth vaccine strain 19 (S19), or znBAZ, and examined for innate immune cell activation. Flow cytometric analysis revealed that znBAZ, but not wt B. abortus nor S19 infection, induces up to a 5-fold increase in the frequency of IFN-γ-producing NK cells in mouse lungs. These NK cells express increased CXCR3 and Ki67, indicating their recruitment and proliferation subsequent to znBAZ infection. Their activation status was augmented noted by the increased NKp46 and granzyme B, but decreased NKG2A expression. Further analysis demonstrated that both lung caspase-1+ inflammatory monocytes and monocyte-derived macrophages secrete chemokines and cytokines responsible for NK cell recruitment and activation. Moreover, neutralizing IL-18, an NK cell-activating cytokine, reduced the znBAZ-induced early NK cell response. NK cell depletion also significantly impaired lung dendritic cell (DC) activation and migration to the lower respiratory lymph nodes (LRLNs). Both lung DC activation and migration to LRLNs were significantly impaired in NK cell-depleted or IFN-γ-/- mice, particularly the CD11b+ and monocytic DC subsets. Furthermore, znBAZ vaccination significantly induced CD8+ T cells, and upon in vivo NK cell depletion, CD8+ T cells were reduced 3-fold compared to isotype-treated mice. In summary, these data show that znBAZ induces lung IFN-γ+ NK cells, which plays a critical role in influencing lung DC activation, migration, and promoting protective CD8+ T cell development.

Brucellosis, caused by Brucella spp., is an important zoonotic disease leading to enormous economic losses in livestock, posing a great threat to public health worldwide. The live attenuated Brucella suis (B. suis) strain S2, a safe and effective vaccine, is widely used in animals in China. However, S2 vaccination in animals may raise debates and concerns in terms of safety to primates, particularly humans. In this study, we used cynomolgus monkey as an animal model to evaluate the safety of the S2 vaccine strain on primates. In addition, we performed transcriptome analysis to determine gene expression profiling on cynomolgus monkeys immunized with the S2 vaccine. Our results suggested that the S2 vaccine was safe for cynomolgus monkeys. The transcriptome analysis identified 663 differentially expressed genes (DEGs), of which 348 were significantly upregulated and 315 were remarkably downregulated. The Gene Ontology (GO) classification and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these DEGs were involved in various biological processes (BPs), including the chemokine signaling pathway, actin cytoskeleton regulation, the defense response, immune system processing, and the type-I interferon signaling pathway. The molecular functions of the DEGs were mainly comprised of 2'-5'-oligoadenylate synthetase activity, double-stranded RNA binding, and actin-binding. Moreover, the cellular components of these DEGs included integrin complex, myosin II complex, and blood microparticle. Our findings alleviate the concerns over the safety of the S2 vaccine on primates and provide a genetic basis for the response from a mammalian host following vaccination with the S2 vaccine.

A novel influenza viral vector based Brucella abortus vaccine (Flu-BA) was introduced for use in cattle in Kazakhstan in 2019. In this study, the safety and efficacy of the vaccine was evaluated in male and female cattle at different ages, and during pregnancy as a part of its registration process. Our data demonstrated that the Flu-BA vaccine was safe after prime or booster vaccination in calves (5–7 months old male and female), heifers (15–17 months old) and cows (6–7 years old) and was not abortogenic in pregnant animals. A mild, localized granuloma was observed at the Flu-BA injection site. Vaccinated animals did not show signs of influenza infection or reduced milk production in dairy cows, and the influenza viral vector (IVV) was not recovered from nasal swabs or milk. Vaccinated animals in all age groups demonstrated increased IgG antibody responses against Brucella Omp16 and L7/L12 proteins with calves demonstrating the greatest increase in humoral responses. Following experimental challenge with B. abortus 544, vaccinates demonstrated greater protection and no signs of clinical disease, including abortion, were observed. The vaccine effectiveness against B. abortus 544 infection was 75, 60 and 60%, respectively, in calves, heifers and adult cows. Brucella were not isolated from calves of vaccinated cattle that were experimentally challenged during pregnancy. Our data suggests that the Flu-BA vaccine is safe and efficacious in cattle, including pregnant animals; and can therefore be administered to cattle of any age.

We compared the effects of two antacid formulations based on sodium bicarbonate and magnesium hydroxide on a Salmonella-delivered oral Brucella live attenuated vaccine. We conducted a series of in vitro and in vivo experiments to investigate the pH buffering capacity, buffering longevity and the effects of these formulations on the survival of Salmonella under neutralized pH conditions and its impact on immune responses. Magnesium hydroxide had a greater, stable and prolonged buffering capacity than sodium bicarbonate and was safer when administered orally. Oral administration of sodium bicarbonate resulted in discomfort as reflected by mouse behavior and mild muscle tremors, whereas mice treated with magnesium hydroxide and PBS were completely normal. Gastric survival studies using BALB/c mice revealed that a higher number of Salmonella reached the intestine when the magnesium hydroxide-based antacid buffer was administrated. Co-administration with attenuated Salmonella secreting Brucella antigens, SodC and Omp19 along with individual antacid formulations, significantly enhanced the antigen-specific protective immune responses against virulent Brucella challenge. Together, our results indicated that the pre vaccinated oral administration of bicarbonate-citric acid or magnesium hydroxide-based neutralizing buffers significantly counteract stomach acidity by maintaining the viability of an oral enteric vaccine formulation.

Background Attenuated Salmonella strain can be used as a vector to transport immunogens to the host antigen-binding sites. Objectives The study aimed to determine the protective efficacy of attenuated Salmonella strain expressing highly conserved Brucella immunogens in goats. Methods Goats were vaccinated with Salmonella vector expressing individually lipoprotein outer-membrane protein 19 (Omp19), Brucella lumazine synthase (BLS), proline racemase subunit A (PrpA), Cu/Zn superoxide dismutase (SOD) at 5 × 109 CFU/mL and challenge of all groups was done at 6 weeks after vaccination. Results Among these vaccines inoculated at 5 × 109 CFU/mL in 1 mL, Omp19 or SOD showed significantly higher serum immunoglobulin G titers at (2, 4, and 6) weeks post-vaccination, compared to the vector control. Interferon-γ production in response to individual antigens was significantly higher in SOD, Omp19, PrpA, and BLS individual groups, compared to that in the vector control (all p < 0.05). Brucella colonization rate at 8 weeks post-challenge showed that most vaccine-treated groups exhibited significantly increased protection by demonstrating reduced numbers of Brucella in tissues collected from vaccinated groups. Real-time polymerase chain reaction revealed that Brucella antigen expression levels were reduced in the spleen, kidney, and parotid lymph node of vaccinated goats, compared to the non-vaccinated goats. Besides, treatment with vaccine expressing individual antigens ameliorated brucellosis-related histopathological lesions. Conclusions These results delineated that BLS, Omp19, PrpA, and SOD proteins achieved a definite level of protection, indicating that Salmonella Typhimurium successfully delivered Brucella antigens, and that individual vaccines could differentially elicit an antigen-specific immune response.

Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×10 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed., This work was partially supported by the FES-CONARE of Costa Rica (www.conare.ac.cr), projects UNA 0009-12, 0248-13, 0504-13, 0505-13 from Universidad Nacional (www.investigacion.una.ac.cr). Grant 803-C0-456, 803-B3-761 Red Temática de brucelosis, Vicerrectoría de Investigación of the University of Costa Rica (www.vinv.ucr.ac.cr). Grant B8762 from Espacio Estudios Avanzados (UCREA) from the University of Costa Rica (www.ucrea.ucr.ac.cr). Bilateral cooperation CSIC-CRUSA Foundation (ref. 2010CR0005)(www.crusa.cr); grants from the Spanish Minister of Economy and Competitiveness (MINECO refs. AGL2010-20247, AGL2011-30453-C04-00, AGL2014-58795-C4-2-R and RTC-2015-3618-1). Fellowship support for The doctoral contract of Ana Zabalza was funded by Public University of Navarra, and the postdoctoral contract of Beatriz San Román was funded by JAE-DOC program of CSIC co-funded by European Social Fund (FSE, European Commission) and MINECO (AGL2011-30453-C04-00) of Spain (www.mineco.gob.es). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The present study employs the Brucella abortus L7/L12 antigen in a Salmonella secretion platform and investigates its ability to induce protective immune responses against wild type challenge in BALB/c mice. The highly conserved L7/L12 open reading frame was PCR amplified from B. abortus and cloned into a prokaryotic expression vector, pJHL65, directly under the beta-lactamase secretory signal. The plasmid constructs pJHL65::L7/L12 was then transformed into an attenuated Salmonella Typhimurium strain, JOL1800 (∆lon, ∆cpxR, ∆asd, and ∆rfaL), and protein secretion was verified by Western blot. Three mice groups were inoculated with either phosphate-buffered saline (PBS), vector-only control, or the vaccine strain secreting L7/L12 antigen. Assessment of humoral and cell-mediated immune responses revealed successful elicitation of Brucella antigen-specific Th1 and Th2 immune responses that were significantly higher than PBS and vector control groups. The immune responses were confirmed by splenocyte proliferation assay, flow cytometry analysis for CD4+ and CD8+ markers, and RT-PCR based cytokine profiling upon restimulation with L7/L12 purified antigen. Results indicate that immunization with Salmonella secreting L7/L12 antigen demonstrated significant enhancement of cell-mediated immune (CMI) responses in immunized mice. The overall effectiveness of the immunization was evaluated by challenging with virulent B. abortus that revealed significant reduction in Brucella colonization in spleen and liver tissues in Salmonella L7/L12 immunized mice. Delivery of Brucella protective antigen L7/L12 using the Salmonella secretion system can effectively accomplish immunogenic advantages of both Salmonella and L7/L12 to derive robust CMI responses and induce humoral immunity to protect against Brucella infection in the mouse model.

Brucella vaccination is one of the most important strategies for controlling brucellosis in livestock. The A19 strain was the effective vaccine used to control brucellosis in China. However, the characteristics of physiological and attenuated virulence of the A19 strain are not investigated in detail. In this study, we compared the phenotypic characteristics of the A19 to the wild-type strain S2308. Virulence test showed that the A19 was significantly attenuated at chronic infection stage in infected mouse model. In growth analysis, the A19 exhibited a quick growth at exponential phase and premature at stationary phase. The inflammatory response of macrophages infected by the A19 was detected using TaqMan qPCR assay, indicating that the inflammatory level of the A19-infected macrophages was higher than that of the S2308 infection. Cell death analysis showed that the A19 was not cytotoxic for macrophages. Cell infection showed that the A19 reduced its ability to invade, survive and traffic within host cells, and the intracellular A19 hardly excludes lysosome-associated marker LAMP-1, suggesting that the A19 can't escape the lysosome degradation within host cells. In further study, the sensitivity test exhibited that the A19 is more sensitive to stress and bactericidal factors than the S2308 strain, Western blot and silver staining analysis exhibited that the A19 has a different expression pattern of OMPs and reduces LPS O-antigen expression relative to the S2308 strain. Those data give us a more detailed understanding about the A19 vaccine strain, which will be beneficial for improvement of current Brucella vaccine and overcoming its defects.

Reproductive failure is the hallmark of brucellosis in animals. An uncommon but important complication in pregnant women who become acutely infected with Brucella melitensis is spontaneous pregnancy loss or vertical transmission to the fetus. Unfortunately, the mechanism behind reproductive failure is still obscure, partially due to the lack of a proper study model. Recently, it was demonstrated that intratracheal (IT) inoculation of nonpregnant guinea pigs would replicate features of clinical disease in humans. To determine if IT inoculation would induce reproductive disease, guinea pigs were infected at mid-gestation and monitored daily for fever and abortions. Fever developed between day 14 to 18 postinoculation, and by 3 weeks postinoculation, 75% of pregnant guinea pigs experienced stillbirths or spontaneous abortions mimicking natural disease. Next, to investigate the guinea pig as a model for evaluating vaccine efficacy during pregnancy, nonpregnant guinea pigs were vaccinated with S19, 16MΔvjbR + Quil-A, or 100 μl PBS + Quil-A (as control). Guinea pigs were bred and vaccinated guinea pigs were challenged at mid-gestation with B. melitensis IT inoculation and monitored for fever and abortions. Vaccination with both vaccines prevented fever and protected against abortion. Together, this study indicates that pregnant guinea pigs are an appropriate animal model to study reproductive disease and offer an improved model to evaluate the ability of vaccine candidates to protect against a serious manifestation of disease.

Brucella, a genus of bacteria that causes brucellosis, infects and threatens domestic animals, and humans in endemic areas. Presently, some live attenuated vaccines of Brucella are used to immunize livestock; however, these vaccines are pathogenic to humans, can provoke abortion when administered to pregnant livestock, and induce antibodies in vaccinated livestock that affect the diagnosis of field infection. It is, therefore, very important for improving the safety and immune protection effects of Brucella vaccine. Currently, recombinant protein-based subunit vaccines are considered promising safe and effective alternatives against brucellosis. Here, we separately expressed the recombinant Omp10-Omp28-L7/L12 proteins of Brucella using eukaryotic and prokaryotic expression systems, which were then used as immunogens for evaluating their immune responses. Taishan Pinus massoniana pollen polysaccharides (TPPPS), an already verified natural adjuvant, was utilized to evaluate the immune conditioning effect on the recombinant proteins. Antibody levels, spleen lymphocyte proliferation, percentages of CD4+ and CD8+ T cells, and cytokine secretion in mice were examined after three successive immunizations. The protective effects against Brucella challenge were also evaluated in mice, and used a live vaccine as a positive control. The results indicated that the immune responses of the recombinant Omp10-Omp28-L7/L12 protein groups were significantly higher than those of the PBS control group. The recombinant Omp10-Omp28-L7/L12 protein expressed in Pichia pastoris (P. pastoris) exhibited a slightly higher expression level and immunogenicity than that expressed in Escherichia coli (E. coli), and the Omp10-Omp28-L7/L12 (P. pastoris) + TPPPS group provided the most pronounced immune effect. The protective results showed that the recombinant Omp10-Omp28-L7/L12 proteins expressed in the two expression systems had significantly better protective effects against Brucella melitensis challenge compared with the negative control, and the addition of TPPPS adjuvant could significantly improve the protective effects of subunit vaccines. However, we also noticed that all of the evaluated subunit vaccines induced less protection than the B. melitensis M5 live vaccine. These results indicate that the combination of recombinant Omp10-Omp28-L7/L12 antigen and TPPPS adjuvant shows potential as an effective brucellosis subunit vaccine, and P. pastoris is a preferred expression system to prepare this recombinant subunit antigen.

Bovine brucellosis, cause by infection with Brucella abortus, causes reproductive failure in cattle, has a major economic impact to producers, and as a zoonoses, it is a disease of public health concern. Characterization of the protective immune response against Brucella infection is important to our understanding of disease pathogenesis and for the development of diagnostic assays and vaccines. Most of the knowledge regarding protection against Brucella comes from studies in the murine model, but less is known about the immune responses in cattle. Assessment of antigen-specific T cell frequency and functional phenotype are critical to understand the immune status of the host, characterize mechanisms of protective immunity and immunopathology, and to predict immune protection. The frequency of circulating T cells specific for a particular pathogen is often very low, making analysis of such responses difficult. Our goal was to develop a flow-cytometry based approach to better track Brucella-specific T cell responses. Using peripheral blood mononuclear cells (PMBC) from Brucella abortus strain RB51-vaccinated cattle, we optimized an in vitro stimulation protocol based on a combination of antigen and pan-T cell stimulation. We then assessed RB51-specific T cell responses by concurrently measuring proliferation and cytokine production using flow-cytometry. This methodology enhances the detection of peripheral, Brucella-specific responses in cattle following RB51 vaccination. This protocol is versatile in that it can be modified to fit other in vitro stimulation systems and additional functional or phenotypic parameters can be added for flow cytometric detection and characterization of antigen-specific T cells.