Brucella Vaccine Research Articles

A Thermosensitive and Degradable Chitin-Based Hydrogel as a Brucellosis Vaccine Adjuvant.

Brucellosis is a zoonotic infectious disease that has long endangered the development of animal husbandry and human health. Currently, vaccination stands as the most efficacious method for preventing and managing brucellosis. Alum, as the most commonly used adjuvant for the brucellosis vaccine, has obvious disadvantages, such as the formation of granulomas and its non-degradability. Therefore, the aims of this study were to prepare an absorbable, injectable, and biocompatible hydroxypropyl chitin (HPCT) thermosensitive hydrogel and to evaluate its immunization efficacy as an adjuvant for Brucella antigens. Specifically, etherification modification of marine natural polysaccharide chitin was carried out to obtain a hydroxypropyl chitin. Rheological studies demonstrated the reversible temperature sensitivity of HPCT hydrogel. Notably, 5 mg/mL of bovine serum albumin can be loaded in HPCT hydrogels and released continuously for more than one week. Furthermore, the L929 cytotoxicity test and in vivo degradation test in rats proved that an HPCT hydrogel had good cytocompatibility and histocompatibility and can be degraded and absorbed in vivo. In mouse functional experiments, as adjuvants for Brucella antigens, an HPCT hydrogel showed better specific antibody expression levels and cytokine (Interleukin-4, Interferon-γ) expression levels than alum. Thus, we believe that HPCT hydrogels hold much promise in the development of adjuvants.

Establishment of an A/T-Rich Specifically MGB Probe digital droplet PCR Assays Based on SNP for Brucella wild strains and vaccine strains

In recent years, immunization with the S2 live-attenuated vaccine has been recognized as the most economical and effective strategy for preventing brucellosis in Inner Mongolia, China. However, there are still challenges related to vaccine toxicity and the inability to distinguish between vaccine immunization and natural infection. Therefore, in this study, we developed a digital droplet polymerase chain reaction (ddPCR) assay based on single-nucleotide polymorphism (SNP) loci to identify wild Brucella strains and S2 vaccine strains. The assay demonstrated excellent linearity (R2> 0.99) with a lower detection limit of 10 copies/µL for both wild and vaccine strains. Additionally, the ddPCR assay outperformed the real-time fluorescent quantitative PCR (qPCR) assay in screening 50 clinical samples. We have established an effective and highly sensitive ddPCR assay for Brucella, providing an efficient method for detecting and differentiating wild strains of Brucella from the S2 vaccine strain.

Development of a novel multi-epitope vaccine for brucellosis prevention

Brucellosis, a zoonotic disease caused by Brucella, presents a significant threat to both animal and human health. In animals, the disease can lead to infertility, miscarriage, and high fever, while in humans, symptoms may include recurrent fever, fatigue, sweating, hepatosplenomegaly, and joint and muscle pain following infection. Treatment often involves long-term antibiotic therapy, placing a substantial psychological and financial burden on patients. While vaccination is crucial for prevention, current animal vaccines have drawbacks such as residual virulence, and a safe and effective human vaccine is lacking. Hence, the development of a vaccine for brucellosis is imperative. In this study, we utilized bioinformatics methods to design a multi-epitope vaccine targeting Brucella. Targeting Heme transporter BhuA and polysaccharide export protein, we identified antigenic epitopes, including six cytotoxic T lymphocyte (CTL) dominant epitopes, six helper T lymphocyte (HTL) dominant epitopes, one conformation B cell dominant epitope, and three linear B cell dominant epitopes. By linking these epitopes with appropriate linkers and incorporating a Toll-like receptor (TLR) agonist (human beta-defensin-2) and an auxiliary peptide (Pan HLA-DR epitopes), we constructed the multi-epitope vaccine (MEV). The MEV demonstrated high antigenicity, non-toxicity, non-allergenicity, non-human homology, stability, and solubility. Molecular docking analysis and molecular dynamics simulations confirmed the interaction and stability of the MEV with receptors (MHCI, MHCII, TLR4). Codon optimization and in silico cloning validated the translation efficiency and successful expression of MEV in Escherichia coli. Immunological simulations further demonstrated the efficacy of MEV in inducing robust immune responses. In conclusion, our findings suggest that the engineered MEVs have the potential to stimulate both humoral and cellular immune responses, offering valuable insights for the future development of safe and efficient Brucella vaccines.

Bactericidal Activity of Serum by Brucella Abortus RB51 Outer Membrane Protein's Combined by Brucella Abortus S99 Lipopolysaccharide Induction.

Brucellosis vaccines are designed to induce cellular immunity. An effective brucellosis vaccine could induce both cellular and humoral immunity. Serum Bactericidal Assay (SBA) is an important method for determining vaccine humoral immunity. This study is the first to observe humoral immunity in brucellosis by SBA. Extracted Brucella abortus (B. abortus) Lipopolysaccharide (LPS) and Outer Membrane Proteins (OMPs) were injected into rabbits. Group 1 was injected with 25 μg of LPS, Group 2 was injected with 50 μg of OMPs, and Group 3 was injected with 1 ml of combined vaccine, 3 times every 2 weeks. The groups were challenged with B. abortus 544 in the second injection. Sera were separated 2 weeks after the last injection. SBA was performed, and each well was streak-cultured into a plate of Brucella agar. A colony count was done for each plate. Results have shown, the third injection of the combined vaccine had the highest titer of , and the efficacy of the vaccine was 87.71%. As a conclusion, the results of this study showed that LPS and OMP's from B. abortus can provide acceptable immunity.

A Review on the Methodology and Use of the Pregnant Mouse Model in the Study of Brucella Reproductive Pathogenesis and Its Abortifacient Effect.

Brucellosis is one of the most common and widespread bacterial zoonoses and is caused by Gram-negative bacteria belonging to the genus Brucella. These organisms are able to infect and replicate within the placenta, resulting in abortion, one of the main clinical signs of brucellosis. Although the mouse model is widely used to study Brucella virulence and, more recently, to evaluate the protection of new vaccines, there is no clear consensus on the experimental conditions (e.g., mouse strains, doses, routes of inoculation, infection/pregnancy time) and the natural host reproducibility of the pregnant mouse model for reproductive brucellosis. This lack of consensus calls for a review that integrates the major findings regarding the effect of Brucella wild-type and vaccine strains infections on mouse pregnancy. We found sufficient evidence on the utility of the pregnant mouse model to study Brucella-induced placentitis and abortion and propose suitable experimental conditions (dose, time of infection) and pregnancy outcome readouts for B. abortus and B. melitensis studies. Finally, we discuss the utility and limitations of the pregnant mouse as a predictive model for the abortifacient effect of live Brucella vaccines.

A novel gluconeogenic route enables efficient use of erythritol in zoonotic Brucella.

Brucellosis is a worldwide extended zoonosis caused by pathogens of the genus Brucella. While most B. abortus, B. melitensis, and B. suis biovars grow slowly in complex media, they multiply intensely in livestock genitals and placenta indicating high metabolic capacities. Mutant analyses in vitro and in infection models emphasize that erythritol (abundant in placenta and genitals) is a preferred substrate of brucellae, and suggest hexoses, pentoses, and gluconeogenic substrates use in host cells. While Brucella sugar and erythritol catabolic pathways are known, growth on 3-4 carbon substrates persists in Fbp- and GlpX-deleted mutants, the canonical gluconeogenic fructose 1,6-bisphosphate (F1,6bP) bisphosphatases. Exploiting the prototrophic and fast-growing properties of B. suis biovar 5, we show that gluconeogenesis requires fructose-bisphosphate aldolase (Fba); the existence of a novel broad substrate bisphosphatase (Bbp) active on sedoheptulose 1,7-bisphosphate (S1,7bP), F1,6bP, and other phosphorylated substrates; that Brucella Fbp unexpectedly acts on S1,7bP and F1,6bP; and that, while active in B. abortus and B. melitensis, GlpX is disabled in B. suis biovar 5. Thus, two Fba-dependent reactions (dihydroxyacetone-phosphate + glyceraldehyde 3-phosphate ⇌ F1,6bP; and dihydroxyacetone-phosphate + erythrose 4-phosphate ⇌ S1,7bP) can, respectively, yield fructose 6-phosphate and sedoheptulose 7-phosphate for classical gluconeogenesis and the Pentose Phosphate Shunt (PPS), the latter reaction opening a new gluconeogenic route. Since erythritol generates the PPS-intermediate erythrose 4-phosphate, and the Fba/Fbp-Bbp route predicts sedoheptulose 7-phosphate generation from erythrose 4-phosphate, we re-examined the erythritol connections with PPS. Growth on erythritol required transaldolase or the Fba/Fbp-Bbp pathway, strongly suggesting that Fba/Fbp-Bbp works as a PPS entry for both erythritol and gluconeogenic substrates in Brucella. We propose that, by increasing erythritol channeling into PPS through these peculiar routes, brucellae proliferate in livestock genitals and placenta in the high numbers that cause abortion and infertility, and make brucellosis highly contagious. These findings could be the basis for developing attenuated brucellosis vaccines safer in pregnant animals.

Fluorescent dual-signal immunosensor combined with machine learning algorithm for accuracy discrimination of natural Brucella infection and vaccination

Brucellosis is a worldwide zoonoses posing a significant threat to humans and animals. The difficulty in distinguishing between Brucella infection and vaccination has led to substantial health risks and economic losses. In this study, we developed a fluorescent dual-signal immunosensor, integrating it with machine learning to solve this problem. Immunogens were selected through structural analysis, followed by the screening of monoclonal antibodies. The antibodies were conjugated with fluorescent microspheres, serving as immunoprobes. Employing a dual-antibody sandwich strategy, we prepared the immunosensor and utilized machine learning to analyze the correlation between multibiomarkers and the status of Brucella infection or vaccine-induced immunity. The immunosensor achieved detection limits of 7.56 pg/mL for IL-1β and 76.61 pg/mL for IL-1Ra, without cross-reactivity with common inflammatory factors. Utilizing the Random Forest algorithm, the combination of the IL-1Ra/IL-1β ratio and RBPT achieved a F1 score of 1.0. Overall, the strategy effectively distinguishes between Brucella-infected and vaccinated subjects, offering valuable applications for brucellosis monitoring.

Contrasting roles for IgM and B-cell MHCII expression in Brucella abortus S19 vaccine-mediated efficacy against B. melitensis infection.

Brucella is a neglected zoonotic pathogen with a worldwide distribution. Our study delves into B-cell effector functions in live vaccine-mediated immunity against brucellosis. Notably, we found antibody production, particularly secretory IgM, confers protection against virulent Brucella melitensis in vaccinated mice, which was associated with complement activation. By contrast, B-cell MHCII expression negatively impacted vaccine efficacy. In addition, B-cell depletion after vaccination, but before the B. melitensis challenge, enhanced protection against infection, suggesting a detrimental B-cell role during the challenge phase. Interestingly, deficiency of T follicular helper cells, which are crucial for aiding germinal center B cells, dampened vaccine efficacy at later stages of challenge independent of antibody production. This study underscores contrasting and phase-dependent roles of B-cell effector functions in vaccine-mediated immunity against Brucella.

Application of alternative identification methods for live tularaemia and brucellosis vaccines

SCIENTIFIC RELEVANCE. The sanctions currently imposed on the Russian Federation requireusing Russian diagnostic products in vaccine quality assessments, as well as searching for alternative testing methods, in particular, serological ones.AIM. This study aimed to demonstrate the possibility of using alternative serological methods, including immunoenzymatic, direct agglutination, and indirect haemagglutination methods, for the identification of tularaemia and brucellosis vaccines in studies and quality assessments. MATERIALS AND METHODS. This study used the established pharmacopoeial reference standard (RS) for the bacterial suspension opacity of 10 international opacity units, the established pharmacopoeial RS for and two commercial batches of the live brucellosis vaccine (Brucella abortus 19 BA), and the established and candidate pharmacopoeial RSs for the live tularaemia vaccine (Francisella tularensis 15 NIIEG). These pharmacopoeial RSs were certified by the Scientific Centre for Expert Evaluation of Medicinal Products. Serological testing used Russian commercial diagnostic products, including dry diagnostic sera (polyvalent brucellosis and tularaemia sera for agglutination tests), a liquid erythrocytic diagnostic preparation of tularaemia immunoglobulin, and enzyme immunoassay (ELISA) diagnostic kits for detecting the causative agents for brucellosis and tularaemia. Statistical analysis involved using Microsoft Excel (P=0.95) for ELISA results and qualitative evaluation for the results of direct agglutination and indirect haemagglutination tests.RESULTS. All the tested batches demonstrated positive results. Live brucellosis vaccine batches showed positive results in the slide agglutination tests, while live tularaemia vaccine batches yielded positive results in the tube agglutination tests. All indirect haemagglutination tests showed haemagglutination in live tularaemia vaccine samples at the same concentration as positive control samples (6.25×106 cells/mL). ELISA tests showed similar optical density values (D)for the two vaccines and positive control samples. Live tularaemia and brucellosis vaccines (undiluted, 1.0×109 cells/mL) had D=2.133±0.273 and D=0.127±0.013, whereas the corresponding control samples had D=1.942±0.056 and D=0.123±0.007, respectively. The results reflected the presence of brucellosis or tularaemia microbes in the samples, confirming the identity of the vaccines.CONCLUSIONS. Serological immunoenzymatic, direct agglutination, and indirect haemagglu tination methods with Russian diagnostic products can be used to identify live brucellosis and tularaemia vaccines during quality assessment. The agglutination method with Russian diagnostic sera can be recommended as an alternative quality assessment method for the identification of live brucellosis and tularaemia vaccines, as this method offers time efficiency, simple visual evaluation of results, and low costs and relatively long shelf lives of diagnostic products. However, ELISA and indirect haemagglutination methods cannot be recommended for this purpose because of their labour-intensive and uneconomical nature. The results of this study may support the introduction of the agglutination method in the regulatory documents for live brucellosis and tularaemia vaccines (as an alternative method).

Ayurvedic Insights into Re-Emerging Human Brucellosis

Brucellosis is a bacterial infection caused by Brucella melintensis. It is one of the most widespread endemic zoonotic diseases present globally, more prevalent in developing countries. It is transmitted to humans either through direct contact with infected animal tissues or fluids or indirectly by ingesting contaminated meat and unpasteurized dairy products. The route of transmission includes mainly mucocutaneous. Farmers, butchers, hunters, and veterinarians are mainly at higher risk of contracting this condition. Mostly the affected individuals exhibit flu-like symptoms, remittent fever, malaise, arthralgia, and weight loss which lasts from a few days to several years. The condition is often misdiagnosed due to nonspecific clinical symptoms, leading to inadequate treatment and prolonged illness leading to severe complications like endocarditis. Currently, there is no vaccine for human brucellosis. The modern management includes administration of antibiotics. The gold standard diagnostic tool is direct bacteriological testing. Ayurvedic understanding of endemic diseases highlights the unique concept of Janapathhodhwamsa proposed by Acharyas. They considered Vayu, Jala, Kala, and Desa as common factors in manifestation of disease. Any alteration in these factors can be understood as imbalance and disease outbreak emerged due to urbanization, lack of hygienic measures in animal husbandry, etc. remaining a public health hazard. Brucellosis is known by its undulant nature which closely resembles the Vishama Jwara in classics which is a Sannipatika type of Jwara. Amapachana, Shodhana, and Samana line of management followed by Rasayana, Satvritta Palana as a preventive line can be adopted to manage Brucellosis.

Parenteral Vaccination with a Live Brucella melitensis Mutant Protects against Wild-Type B. melitensis 16M Challenge.

Susceptibility to brucellosis remains prevalent, even in herds vaccinated with conventional vaccines. Efforts are underway to develop an improved brucellosis vaccine, and possibly a universal vaccine, given that Brucella species are highly homologous. To this end, two B. melitensis mutants were developed, znBM-lacZ (znBMZ) and znBM-mCherry (znBM-mC), and were tested for their ability to confer systemic immunity against virulent B. melitensis challenge. To assess the extent of their attenuation, bone-marrow-derived macrophages and human TF-1 myeloid cells were infected with both mutants, and the inability to replicate within these cells was noted. Mice infected with varying doses of znBM-mC cleared the brucellae within 6-10 weeks. To test for efficacy against systemic disease, groups of mice were vaccinated once by the intraperitoneal route with either znBMZ or B. abortus S19 vaccine. Relative to the PBS-dosed mice, znBMZ vaccination greatly reduced splenic brucellae colonization by ~25,000-fold compared to 700-fold for S19-vaccinated mice. Not surprisingly, both znBMZ and S19 strains induced IFN-γ+ CD4+ T cells, yet only znBMZ induced IFN-γ+ CD8+ T cells. While both strains induced CD4+ effector memory T cells (Tems), only znBMZ induced CD8+ Tems. Thus, these results show that the described znBM mutants are safe, able to elicit CD4+ and CD8+ T cell immunity without a boost, and highly effective, rendering them promising vaccine candidates for livestock.

Breeding and Healthcare Practices Followed by the Rajbanshi Dairy Farmers in Coochbehar District of West Bengal

Breeding and healthcare management play a significant role in utilizing the full potential of dairy animals. Constant breeding and healthcare development are essential to provide better conditions and improve dairy production and welfare. This study explores the breeding and healthcare management practices adopted by Rajbanshi dairy owners in West Bengal’s Coochbehar district. Utilizing a pre-tested interview schedule, data was collected and subsequently analyzed in terms of frequency and percentage. The findings shed light on key patterns and practices, contributing to a better understanding of dairy management within this specific community. A strong preference (77.00%) for crossbreed cattle emerges in breeding practices meanwhile, heat detection predominantly relies on bellowing and mucus discharge (86.00%) and the pregnancy diagnosis was mostly done by Prani Bondhu (88.64 %). Within healthcare practices, dairy farm families vaccinated their animals by 98.50%, 19.50%, 16.50% and 52.50% of their animals with FMD, HS, BQ and Brucellosis vaccines respectively. Half of the dairy farm families (49.50%) followed deworming practices occasionally. The findings highlight the need for specific training within the dairy farming community on reproductive techniques and disease prevention methods. Also, promoting hygienic practices in animal housing and expanding access to veterinary care can improve overall animal health and farm sustainability.

Immunoinformatics analysis of Brucella melitensis to approach a suitable vaccine against brucellosis

BackgroundBrucellosis caused by B. melitensis is one of the most important common diseases between humans and livestock. Currently, live attenuated vaccines are used for this disease, which causes many problems, and unfortunately, there is no effective vaccine for human brucellosis. The aim of our research was to design a recombinant vaccine containing potential immunogenic epitopes against B. melitensis. MethodsIn this study, using immunoinformatics approaches, 3 antigens Omp31, Omp25, and Omp28 were identified and the amino acid sequence of the selected antigens was determined in NCBI. Signal peptides were predicted by SignaIP-5.0 server. To predict B-cell epitopes from ABCpred and Bcepred servers, to predict MHC-I epitopes from RANKPEP and SYFPEITHI servers, to predict MHC-II epitopes from RANKPEP and MHCPred servers, and to predict CTL epitopes were used from the CTLPred server. Potentially immunogenic final epitopes were joined by flexible linkers. Finally, allergenicity (AllerTOP 2.0 server), antigenicity (Vaxijen server), physicochemical properties (ProtParam server), solubility (Protein-sol server), secondary (PSIPRED and GRO4 servers) and tertiary structure (I-TASSER server), refinement (GalaxyWEB server), validation (ProSA-web, Molprobity, and ERRAT servers), and optimization of the codon sequence (JCat server) of the structure of the multi-epitope vaccine were analyzed. ResultsThe analysis of immunoinformatics tools showed that the designed vaccine has high quality, acceptable physicochemical properties, and can induce humoral and cellular immune responses against B. melitensis bacteria. In addition, the high expression level of recombinant antigens in the E. coli host was observed through in silico simulation. ConclusionAccording to the results in silico, the designed vaccine can be a suitable candidate to fight brucellosis and in vitro and in vivo studies are needed to evaluate the research of this study.

One-step preparation of a self-assembled bioconjugate nanovaccine against Brucella

ABSTRACT Brucellosis, caused by Brucella, is a severe zoonosis, and the current Brucella live attenuated vaccine cannot be used in humans due to major safety risks. Although polysaccharide antigens can be used to prepare the Brucella vaccine, their lower immunogenicity limits them from producing efficient and broad protection. In this study, we produced a high-performance bioconjugate nanovaccine against different species of Brucella by introducing a self-assembly nanoparticle platform and an O-linked glycosylation system into Yersinia enterocolitica serotype O:9, which has an O-polysaccharide composed of the same unit as Brucella. After successfully preparing the vaccine and confirming its stability, we subsequently demonstrated the safety of the vaccine in mice by high-dose immunization. Then, by a series of mouse experiments, we found that the nanovaccine greatly promoted antibody responses. In particular, the increase of IgG2a was more obvious than that of IgG1. Most importantly, this nanovaccine could provide cross-protection against B. abortus, B. melitensis, and B. suis strains by lethal dose challenged models, and could improve the clearance of B. melitensis, the most common pathogenic species in human brucellosis, by non-lethal dose infection. Overall, for the first time, we biocoupled polysaccharide antigens with nano carriers to prepare a Brucella vaccine, which showed pronounced and extensive protective effects in mice. Thus, we provided a potential candidate vaccine and a new direction for Brucella vaccine design.

Brucella melitensis Vaccines: A Systematic Review

Background: Brucella melitensis is recognized as one of the predominant zoonotic pathogens globally. Live-attenuated vaccine Rev 1 is currently the most effective vaccine for controlling B. melitensis in small ruminants. While Brucella inactivated, nanoparticle, and subunit vaccines are less effective and require multiple doses, live-attenuated vaccines are less expensive and more efficacious. Several drawbacks are associated with the administration of current attenuated B. melitensis vaccines, including interference with serological diagnostic tests, inducing abortion in pregnant animals, shedding in milk, and zoonotic infections in humans. In this systematic review, we summarize the current literature (1970–2022) on B. melitensis vaccines and review their advantages and disadvantages in order to support the rationale for a need for new or improved small ruminant brucellosis vaccines. Methods: A systematic search was carried out in Web of Science, CAB Abstracts, and PubMed. The original articles describing the B. melitensis vaccines were included. Review articles, articles not published in English, articles that did not offer full text, editorials, correspondences, case reports, case series, diagnostic tests, duplicate publications, and other Brucella vaccines (e.g., B. abortus and B. suis) were excluded. Results: Out of 3700 studies, we identified 18 articles that evaluated B. meltensis vaccines, including recombinant B. melitensis strains (16MΔhfq, 16MΔTcfSR, M5-90ΔmanB, LVM31, M5-90ΔvjbR, 16MΔmucR, ΔznuA, M5-90Δpgm, M5-90ΔwboA), live B. melitensis strain (Rev 1), nanoparticle vaccines (B. melitensis 16M, B. melitensis OMP 31, FliC protein—Mannosylated Chitosan Nanoparticles (FliC and FliC-MCN), B. melitensis and B. abortus combined, and B. melitensis 16M nanoparticles combined with oligopolysaccharide), subunit vaccines (outer membrane vesicles or outer membrane proteins), and a DNA vaccine based on B. melitensis outer membrane proteins (Omp25 and Omp31). The results from these studies revealed that these vaccines can induce humoral and cellular responses and reduce macrophage survival. However, most of these vaccines were evaluated only in murine models, which may not accurately reflect how they work in natural hosts. Conclusions: The high prevalence of B. melitensis in humans and animals remains an issue in many parts of the world. Human brucellosis can be prevented by controlling brucellosis in livestock using vaccination and test-and-removal strategies. Prospective vaccines have limitations, including interference with serodiagnostics after vaccination, virulence in humans and animals, the requirement of booster vaccinations, and insufficient efficacy in preventing infection or abortion. Moreover, most of these vaccines have been assessed in mice models, which have failed to predict immunogenicity or efficacy in natural hosts. Because of these limitations and the re-emergence of B. melitensis worldwide with a high incidence of human infection, our review suggests a need for additional research into the molecular pathology and immunological properties of B. melitensis infection and the identification of protective epitopes or genes that would allow for the development of improved vaccines for small ruminants.

Design of a multi-epitope vaccine against brucellosis fused to IgG-fc by an immunoinformatics approach.

Brucella, a type of intracellular Gram-negative bacterium, has unique features and acts as a zoonotic pathogen. It can lead to abortion and infertility in animals. Eliminating brucellosis becomes very challenging once it spreads among both humans and animals, putting a heavy burden on livestock and people worldwide. Given the increasing spread of brucellosis, it is crucial to develop improved vaccines for susceptible animals to reduce the disease's impact. In this study, we effectively used an immunoinformatics approach with advanced computer software to carefully identify and analyze important antigenic parts of Brucella abortus. Subsequently, we skillfully designed chimeric peptides to enhance the vaccine's strength and effectiveness. We used computer programs to find four important parts of the Brucella bacteria that our immune system recognizes. Then, we carefully looked for eight parts that are recognized by a type of white blood cell called cytotoxic T cells, six parts recognized by T helper cells, and four parts recognized by B cells. We connected these parts together using a special link, creating a strong new vaccine. To make the vaccine even better, we added some extra parts called molecular adjuvants. These included something called human β-defensins 3 (hBD-3) that we found in a database, and another part that helps the immune system called PADRE. We attached these extra parts to the beginning of the vaccine. In a new and clever way, we made the vaccine even stronger by attaching a part from a mouse's immune system to the end of it. This created a new kind of vaccine called MEV-Fc. We used advanced computer methods to study how well the MEV-Fc vaccine interacts with certain receptors in the body (TLR-2 and TLR-4). In the end, Immunosimulation predictions showed that the MEV-Fc vaccine can make the immune system respond strongly, both in terms of cells and antibodies. In summary, our results provide novel insights for the development of Brucella vaccines. Although further laboratory experiments are required to assess its protective effect.

Immunization of BALB/c mice with BAB1-0278: An initial investigation of a novel potential vaccine for brucellosis based on Lactococcus Lactis vector

The gram-negative intracellular bacterium Brucella abortus causes bovine brucellosis, a zoonotic disease that costs a lot of money. This work developed a vector vaccine against brucellosis utilizing recombinant L. lactis expressing Brucella outer membrane protein BAB1-0278. Gene sequences were obtained from GenBank. The proteins' immunogenicity was tested with Vaxijen. The target vector was converted into L. lactis after enzymatic digestion and PCR validated the BAB1-0278 gene cloning in the pNZ8148 vector. The target protein was extracted using a Ni-NTA column and confirmed using SDS-PAGE and western blot. After vaccination with the target vaccine, the expression of IgG subclasses was evaluated by the ELISA method. Cytokine production was also measured by the qPCR method in the small intestine and spleen. Lymphocyte proliferation and innate immune response (NLR, CRP, and PLR) were also assessed. Finally, after the challenge test, the spleen tissue was examined by H&E staining. BAB1-0278 was chosen because of its antigenicity score of 0.5614. A 237-bp gene fragment was discovered using enzymatic digestion and PCR. The presence of a 13 kDa protein band was confirmed by SDS-PAGE and western blot. In comparison to the PBS group, mice given the L. lactis-pNZ8148-BAB1-0278-Usp45 vaccine 14 days after priming had substantially greater levels of total IgG, IgG1, and IgG2a (P < 0.001). Also, the production of cytokines (IFN-γ, TNFα, IL-4, and IL-10) indicating cellular immunity increased compared to the control group (P < 0.001). The target group had a lower inflammatory response, morphological impairment, alveolar edema, and lymphocyte infiltration. An efficient probiotic-based oral brucellosis vaccination was created. These studies have proven that the recommended immunization gives the best protection, which supports its promotion.

A novel Galleria mellonella experimental model for zoonotic pathogen Brucella

ABSTRACT Brucellosis is a major threat to public health and animal husbandry. Several in vivo vertebrate models, such as mice, guinea pigs, and nonhuman primates, have been used to study Brucella pathogenesis, bacteria-host interactions, and vaccine efficacy. However, these models have limitations whereas the invertebrate Galleria mellonella model is a cost-effective and ethical alternative. The aim of the present study was to examine the invertebrate G. mellonella as an in vivo infection model for Brucella. Infection assays were employed to validate the fitness of the larval model for Brucella infection and virulence evaluation. The protective efficacy of immune sera was evaluated by pre-incubated with a lethal dose of bacteria before infection. The consistency between the mouse model and the larval model was confirmed by assessing the protective efficacy of two Brucella vaccine strains. The results show that G. mellonella could be infected by Brucella strains, in a dose- and temperature-dependent way. Moreover, this larval model can effectively evaluate the virulence of Brucella strains in a manner consistent with that of mammalian infection models. Importantly, this model can assess the protective efficacy of vaccine immune sera within a day. Further investigation implied that haemolymph played a crucial role in the protective efficacy of immune sera. In conclusion, G. mellonella could serve as a quick, efficient, and reliable model for evaluating the virulence of Brucella strains and efficacy of immune sera in an ethical manner.

Brucellae as resilient intracellular pathogens: epidemiology, host-pathogen interaction, recent genomics and proteomics approaches, and future perspectives.

Brucellosis is considered one of the most hazardous zoonotic diseases all over the world. It causes formidable economic losses in developed and developing countries. Despite the significant attempts to get rid of Brucella pathogens in many parts of the world, the disease continues to spread widely. Recently, many attempts proved to be effective for the prevention and control of highly contagious bovine brucellosis, which could be followed by others to achieve a prosperous future without rampant Brucella pathogens. In this study, the updated view for worldwide Brucella distribution, possible predisposing factors for emerging Brucella pathogens, immune response and different types of Brucella vaccines, genomics and proteomics approaches incorporated recently in the field of brucellosis, and future perspectives for prevention and control of bovine brucellosis have been discussed comprehensively. So, the current study will be used as a guide for researchers in planning their future work, which will pave the way for a new world without these highly contagious pathogens that have been infecting and threatening the health of humans and terrestrial animals.

Pathogenesis, clinical manifestations, treatment and prevention of brucellosis in humans and animals

Brucella genus is Gram-negative coccobacilli that cause a zoonotic disease called brucellosis in humans and animals. 12 species have been identified for Brucella, among which four species, Brucella melitensis, Brucella suis, Brucella abortus, and Brucella canis, commonly cause disease in humans and animals. Brucellosis is transmissible and there are different types of transmission such as animal-to-human (dairy and meat consumption), human-to-human (placenta, breastfeeding, sexual intercourse, blood transfusion, and bone marrow transplantation), and animal-to-animal (aerosol-containing Brucella, placenta and sexual intercourse). Due to having virulence factors such as type IV secretion system (T4SS), superoxide dismutase enzyme, BvrS/BvrR two-component system, LPS with O chain, and cyclic beta-glucan, Brucella can survive and replicate intracellularly and cause disease. Fever, excessive sweating, malaise, myalgia, arthralgia, loss of appetite, weight loss, hepatosplenomegaly, spondylitis, and arthritis are the clinical manifestations of brucellosis in humans. In animals, complications such as abortion, weak calf birth, reduced fertility, endometritis, reduced milk production, orchitis, epididymis, and hygroma may be observed. To diagnose brucellosis, a direct method such as culture or indirect and serological methods such as the Rose Bengal test (RBT), tube standard agglutination test (SAT), 2-mercaptoethanol test, Coombs test, and ELISA are used. According to the condition of the patient, the combination of doxycycline with streptomycin, gentamicin, or rifampin is used to treat brucellosis. While animals with brucellosis are not treated and are slaughtered. No vaccine against brucellosis has been approved for humans yet. But for animals, S19, Rev.1, S2, RB51, and SR82 vaccines are used for prevention. Development and approval of brucellosis vaccine for humans, wider use of animal vaccines, and timely diagnosis of suspected cases will help to better control brucellosis and prevent economic consequences.