A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

The immunostimulatory roles of gold nanoparticles in immunization and vaccination against Brucella abortus antigens.

One effective antigen carrier proposed for use in immunization and vaccination is gold nanoparticles. Prior work has shown that gold nanoparticles themselves have adjuvant properties. Currently, gold nanoparticles are used to design new diagnostic tests and vaccines against viral, bacterial, and parasitic infections. We investigated the use of gold nanoparticles as immunomodulators in immunization and vaccination with an antigen isolated from Brucella abortus. Gold nanoparticles with a diameter of 15 nm were synthesized for immunization of animals and were then conjugated to the isolated antigen. The conjugates were used to immunize white BALB/c mice. As a result, high-titer (1:10240) antibodies were produced. The respiratory and proliferative activities of immune cells were increased, as were the serum interleukin concentrations. The minimum antigen amount detected with the produced antibodies was âˆ¼ 0.5 pg. The mice immunized with gold nanoparticles complexed with the B. abortus antigen were more resistant to B. abortus strain 82 than were the mice immunized through other schemes. This fact indicates that animal immunization with this conjugate enhances the effectiveness of the immune response. The results of this study are expected to be used in further work to examine the protective effect of gold nanoparticles complexed with the B. abortus antigen on immunized animals and to develop test systems for diagnosing brucellosis in the laboratory and in the field.

Exosomes released by Brucella-infected macrophages inhibit the intracellular survival of Brucella by promoting the polarization of M1 macrophages.

Exosomes, membrane vesicles released extracellularly from cells, contain nucleic acids, proteins, lipids and other components, allowing the transfer of material information between cells. Recent studies reported the role of exosomes in pathogenic microbial infection and host immune mechanisms. Brucella-invasive bodies can survive in host cells for a long time and cause chronic infection, which causes tissue damage. Whether exosomes are involved in host anti-Brucella congenital immune responses has not been reported. Here, we extracted and identified exosomes secreted by Brucella melitensis M5 (Exo-M5)-infected macrophages, and performed in vivo and in vitro studies to examine the effects of exosomes carrying antigen on the polarization of macrophages and immune activation. Exo-M5 promoted the polarization of M1 macrophages, which induced the significant secretion of M1 cytokines (tumour necrosis factor-α and interferon-γ) through NF-κB signalling pathways and inhibited the secretion of M2 cytokines (IL-10), thereby inhibiting the intracellular survival of Brucella. Exo-M5 activated innate immunity and promoted the release of IgG2a antibodies that protected mice from Brucella infection and reduced the parasitaemia of Brucella in the spleen. Furthermore, Exo-M5 contained Brucella antigen components, including Omp31 and OmpA. These results demonstrated that exosomes have an important role in immune responses against Brucella, which might help elucidate the mechanisms of host immunity against Brucella infection and aid the search for Brucella biomarkers and the development of new vaccine candidates.

Immunoinformatic-guided designing of multi-epitope vaccine construct against Brucella Suis 1300.

Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to survive and multiply in a phagocytic environment and to acquire resistance under hostile conditions thus becoming a threat globally. Antibiotic resistance is posing a substantial public health threat, hence there is an unmet and urgent clinical need for immune-based non-antibiotic methods to treat brucellosis. Hence, we aimed to explore the whole proteome of Brucella suis to predict antigenic proteins as a vaccine target and designed a novel chimeric vaccine (multi-epitope vaccine) through subtractive genomics-based reverse vaccinology approaches. The applied subsequent hierarchical shortlisting resulted in the identification of Multidrug efflux Resistance-nodulation-division (RND) transporter outer membrane subunit (gene BepC) that may act as a potential vaccine target. T-cell and B-cell epitopes have been predicted from target proteins using a number of immunoinformatic methods. Six MHC I, ten MHC II, and four B-cell epitopes were used to create a 324-amino-acid MEV construct, which was coupled with appropriate linkers and adjuvant. To boost the immunological response to the vaccine, the vaccine was combined with the TLR4 agonist HBHA protein. The MEV structure predicted was found to be highly antigenic, non-toxic, non-allergenic, flexible, stable, and soluble. To confirm the interactions with the receptors, a molecular docking simulation of the MEV was done using the human TLR4 (toll-like receptor 4) and HLAs. The stability and binding of the MEV-docked complexes with TLR4 were assessed using molecular dynamics (MD) simulation. Finally, MEV was reverse translated, its cDNA structure was evaluated, and then, in silico cloning into an E. coli expression host was conducted to promote maximum vaccine protein production with appropriate post-translational modifications. These comprehensive computer calculations backed up the efficacy of the suggested MEV in protecting against B. suis infections. However, more experimental validations are needed to adequately assess the vaccine candidate's potential. HIGHLIGHTS: • Subtractive genomic analysis and reverse vaccinology for the prioritization of novel vaccine target • Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy, and structural-based studies • Molecular docking simulation method to rank based vaccine candidate and understand their binding modes.

Primary and memory immune responses against rough Brucella canis are less robust compared to smooth B. abortus and B. melitensis following intratracheal infection in mice.

Brucella canis is the cause of canine brucellosis, a globally distributed, zoonotic pathogen which primarily causes disease in dogs. B. canis is unique amongst the zoonotic Brucella spp. with its rough lipopolysaccharide, a trait typically associated with attenuation in gram-negative bacteria. Unfortunately, no vaccine is available against B. canis, and vaccine development is hampered by a limited understanding of the immune response required to combat it and the course of infection following a physiologically relevant, mucosal route of inoculation. To address these concerns and analyze the impact of the rough phenotype on the immune response, we infected mice intratracheally with rough B. canis or smooth B. melitensis or B. abortus. Bacterial colonization and histologic lesions were assessed in systemic target organs as well as locally in the lungs and draining mediastinal lymph node. Mice were also reinfected with Brucella following antibiotic treatment and cytokine production by T lymphocytes in the lung and spleen was assessed by flow cytometry to investigate the memory immune response. Despite its rough phenotype, B. canis established a persistent infection at the same level of colonization as the smooth strains. However, B. canis induced significantly less granulomatous inflammation in the spleen as well as a lack of bronchial-associated lymphoid tissue (BALT) hyperplasia in the lungs. These differences coincided with increased IL-10 and decreased IFN-γ in the spleen of B. canis-infected mice. Previous exposure to all Brucella strains provided protection against colonization following secondary challenge, although induction of IFN-γ by T lymphocytes was seen only in the lungs during B. canis infection while the smooth strains induced this cytokine in the spleen as well. Neither Brucella strain induced significant polyfunctional T lymphocytes, a potential immunomodulatory mechanism that appears to be independent of lipopolysaccharide phenotype.

A Brucella Omp16 Conditional Deletion Strain Is Attenuated in BALB/c Mice.

Brucella spp. are facultative intracellular pathogens that invade, survive and proliferate in numerous phagocytic and non-phagocytic cell types, thereby leading to human and animal brucellosis. Outer membrane proteins (Omps) are major immunogenic and protective antigens that are implicated in Brucella virulence. A strain deleted of the omp16 gene has not been obtained which suggests that the Omp16 protein is vital for Brucella survival. Nevertheless, we previously constructed an omp16 conditional deletion strain of Brucella, ΔOmp16. Here, the virulence and immune response elicted by this strain were assessed in a mouse model of infection. Splenomegaly was significantly reduced at two weeks post-infection in ΔOmp16-infected mice compared to infection with the parental strain. The bacterial load in the spleen also was significantly decreased at this post-infection time point in ΔOmp16-infected mice. Histopathological changes in the spleen were observed via hematoxylineosin staining and microscopic examination which showed that infection with the ΔOmp16 strain alleviated spleen histopathological alterations compared to mice infected with the parental strain. Moreover, the levels of humoral and cellular immunity were similar in both ΔOmp16-infected mice and parental strain-infected mice. The results overall show that the virulence of ΔOmp16 is attenuated markedly, but that the immune responses mediated by the deletion and parental strains in mice are indistinguishable. The data provide important insights that illuminate the pathogenic strategies adopted by Brucella.

Aconitate decarboxylase 1 participates in the control of pulmonary Brucella infection in mice.

Brucellosis is one of the most widespread bacterial zoonoses worldwide. Here, our aim was to identify the effector mechanisms controlling the early stages of intranasal infection with Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main cells infected in the lungs. Using RNA sequencing, we identified the aconitate decarboxylase 1 gene (Acod1; also known as Immune responsive gene 1), as one of the genes most upregulated in murine AMs in response to B. melitensis infection at 24 hours post-infection. Upregulation of Acod1 was confirmed by RT-qPCR in lungs infected with B. melitensis and B. abortus. We observed that Acod1-/- C57BL/6 mice display a higher bacterial load in their lungs than wild-type (wt) mice following B. melitensis or B. abortus infection, demonstrating that Acod1 participates in the control of pulmonary Brucella infection. The ACOD1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite in the Krebs cycle, into itaconate. Dimethyl itaconate (DMI), a chemically-modified membrane permeable form of itaconate, has a dose-dependent inhibitory effect on Brucella growth in vitro. Interestingly, structural analysis suggests the binding of itaconate into the binding site of B. abortus isocitrate lyase. DMI does not inhibit multiplication of the isocitrate lyase deletion mutant ΔaceA B. abortus in vitro. Finally, we observed that, unlike the wt strain, the ΔaceA B. abortus strain multiplies similarly in wt and Acod1-/- C57BL/6 mice. These data suggest that bacterial isocitrate lyase might be a target of itaconate in AMs.

Hybrid recombinant Omp 22, 25, and 31 immunodominant epitopes can be used for serodiagnosis of brucellosis.

Brucellosis is a well-known infectious disease in most parts of the world, especially in developing countries, common between humans and animals. Brucellosis is diagnosed by serological tests based on lipopolysaccharides (LPSs), which are bacterial cell wall antigens, and due to the similarities between LPSs antigens of some gram-negative bacterias, false-positive responses are inevitable. Alternatively, Outer membrane proteins (Omps), as antigenic conserved membrane proteins, can be used to diagnose brucellosis instead of LPS antigens. In this study, by using bioinformatics tools, linear B-cell epitopes were selected from Omp22, Omp25, and Omp31 antigens and fused with the rigid KP linker (K = Lysine, P=Proline). Designed gene cassette was cloned into pET-28a (+) vector and expressed recombinant protein was purified using Ni-NTA chromatography column and was confirmed with Poly-Histidine-HRP antibody. Finally, recombinant protein's seroreactivity with serum samples from 37 patients and 27 healthy individuals was evaluated by western blotting and enzyme-linked immunosorbent assay (ELISA) methods. Western blotting results showed high reactivity of the recombinant protein with serum samples of Brucella infected patients. ELISA results were analyzed using the receiver operating curve (ROC). Optical density cut-off point, accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Youden index J for recombinant protein were > 0.809, 84.37%,83.78%,88.89%,88.57%, 79.31% and 0.72 respectively. Western blotting and ELISA results showed that our recombinant protein has good sensitivity and specificity for the diagnosis of brucellosis.

Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune-related signaling pathways and gut microbiota.

Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92 ± 70.6074 vs 263.10 ± 34.60 pg/mL, P < .05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.

A Brucella effector modulates the Arf6-Rab8a GTPase cascade to promote intravacuolar replication.

Remodeling of host cellular membrane transport pathways is a common pathogenic trait of many intracellular microbes that is essential to their intravacuolar life cycle and proliferation. The bacterium Brucella abortus generates a host endoplasmic reticulum-derived vacuole (rBCV) that supports its intracellular growth, via VirB Type IV secretion system-mediated delivery of effector proteins, whose functions and mode of action are mostly unknown. Here, we show that the effector BspF specifically promotes Brucella replication within rBCVs by interfering with vesicular transport between the trans-Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN, and interacted with the Arf6 GTPase-activating Protein (GAP) ACAP1 to dysregulate Arf6-/Rab8a-dependent transport within the recycling endosome, which resulted in accretion of TGN-associated vesicles by rBCVs and enhanced bacterial growth. Altogether, these findings provide mechanistic insight into bacterial modulation of membrane transport used to promote their own proliferation within intracellular vacuoles.

Mucosal Vaccination Primes NK Cell-Dependent Development of CD8+ T Cells Against Pulmonary Brucella Infection.

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.

Soroprevalência de brucelose em suínos de granjas comerciais e em criatórios de subsistência / Seroprevalence of brucellosis in farm animals and in subsistence breeders

A suinocultura industrial compreende o conjunto de produtores que incorporam os avanços tecnológicos em genética, nutrição, sanidade e demais aspectos produtivos, enquanto a suinocultura de subsistência desempenha um papel importante para a alimentação humana, sendo a criação de suínos em criatórios amplamente difundida entre os pequenos proprietários de terra ou em assentamentos. Este trabalho teve como objetivo avaliar a pesquisa de anticorpos contra Brucella sp. nos suínos de granjas comerciais, criatórios de subsistência e javalis asselvajados. Foram visitados 32 criatórios de suínos domésticos localizados na região sul de Mato Grosso do Sul. Considerando-se os fenótipos, verificou-se que os sinais indicativos de contato têm influência do município onde está situada a propriedade (χ2=8.8594, p=0,0029), sendo que o município de Deodápolis tem uma chance de ocorrência mais elevada (OR=13,00;IC95%:2,12-79,59). Através da detecção fenotípicaobserva-sea presença de animais híbridos nas propriedades analisadas, e evidencia-se assim que os animais da propriedade em algum momento entraram em contato com suínos asselvajados, os quais podem ser responsáveis pela disseminação de diversas patologias. A ausência da detecção de anticorpos anti-Brucella sp. em suínos de criatórios sugere que a bactéria não é circulante nas populações de javalis nas áreas de estudo.

Industrial pig farming comprises the group of producers that incorporate technological advances in genetics, nutrition, health and other productive aspects, while subsistence pig farming plays an important role for human nutrition, with the creation of pigs in farms being widely spread among small farms. landowners or settlements. This study aimed to evaluate the search for antibodies against Brucella sp. pigs on commercial farms, livestock farms and wild boars. Were visited 32 domestic swine farms located in the southern region of Mato Grosso do Sul. Considering the phenotypes, it was found that the indicative signs of contact have influence from the municipality where the property is located (χ2=8.8594, p=0.0029) , and the municipality of Deodápolis has a higher chance of occurrence (OR=13,00;IC95%:2,12-79,59). Through the phenotypic detection, the presence of hybrid animals in the analyzed properties is clearly observed, and it is evident that the animals of the property at some point came into contact with pigs, which may be responsible for the spread of several pathogens. The absence of detection of anti-Brucella sp. in breeding pigs it suggests that the bacteria is not circulating in wild boar populations in the study areas.

In Vitro and In Vivo IFN-γ and IL-10 Measurement in Experimental Brucella abortus Biotype 1 Infection in Sprague-Dawley Rats.

The immune response to Brucella abortus mainly depends on antigen-specific T cell activation, CD4+ and CD8+ T cells, and Brucella-specific humoral response. Protective immune response against Brucella infection has not been performed in the Sprague-Dawley (SD) rat model. We measured bacterial kinetics in addition to in vivo and in vitro interferon gamma (IFN-γ) and interleukin-10 (IL-10) production against crude Brucella protein in the SD rats at different days of postinfection with B. abortus biotype 1 by indirect enzyme-linked immunosorbent assay. Forty SD rats were inoculated intraperitoneally with 0.1 mL sterile injectable pyrogen-free solution containing 1 × 1010 colony-forming units/mL of B. abortus biotype 1 obtained from cattle in Korea. Four rats were used as uninfected control. Serum IFN-γ level at 3 and 7 days postinfection were significantly higher (p > 0.001) compared with the IL-10 level. On the contrary, serum IL-10 levels were observed significantly higher at 21 and 28 days postinfection compared with the serum IFN-γ levels (p < 0.001). The production of IFN-γ by spleen cells was significantly higher at 7 and 14 days postinfection compared with IL-10 (p < 0.001). On the contrary, IL-10 productions were found to be significantly higher at 21, 28, 35, and 42 days postinfection compared with IFN-γ (p < 0.001). The presence of B. abortus in blood was marked till 5 weeks of infection, throughout the experiment in case of spleen, and no bacteria were isolated from the kidney and liver at 6 weeks postinfection. The in vivo and in vitro IFN-γ and IL-10 measurement in our study reported that B. abortus infection in rats primarily educe T helper (Th)1-dominant immune response in acute infection accompanied by Th2-dominant immune response in chronic infection.

STING regulates metabolic reprogramming in macrophages via HIF-1α during Brucella infection.

Macrophages metabolic reprogramming in response to microbial insults is a major determinant of pathogen growth or containment. Here, we reveal a distinct mechanism by which stimulator of interferon genes (STING), a cytosolic sensor that regulates innate immune responses, contributes to an inflammatory M1-like macrophage profile upon Brucella abortus infection. This metabolic reprogramming is induced by STING-dependent stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), a global regulator of cellular metabolism and innate immune cell functions. HIF-1α stabilization reduces oxidative phosphorylation and increases glycolysis during infection with B. abortus and, likewise, enhances nitric oxide production, inflammasome activation and IL-1ß release in infected macrophages. Furthermore, the induction of this inflammatory profile participates in the control of bacterial replication since absence of HIF-1α renders mice more susceptible to B. abortus infection. Mechanistically, activation of STING by B. abortus infection drives the production of mitochondrial reactive oxygen species (mROS) that ultimately influences HIF-1α stabilization. Moreover, STING increases the intracellular succinate concentration in infected macrophages, and succinate pretreatment induces HIF-1α stabilization and IL-1ß release independently of its cognate receptor GPR91. Collectively, these data demonstrate a pivotal mechanism in the immunometabolic regulation of macrophages during B. abortus infection that is orchestrated by STING via HIF-1α pathway and highlight the metabolic reprogramming of macrophages as a potential treatment strategy for bacterial infections.

Characterization of innate immune response to Brucella melitensis infection in goats with permissive or restrictive phenotype for Brucella intramacrophagic growth.

Caprine brucellosis is a chronic, world-wide distributed disease which causes reproductive failure in goats and Brucella melitensis, its causative agent, bears a great zoonotic potential. There is evidence suggesting that some cattle and pigs have an innate ability to resist Brucella infection, but this has not yet been investigated in goats. In this study, we compared caprine macrophages that exhibit extreme restriction and permissiveness to B. melitensis' intracellular growth in vitro. Monocyte derived macrophages (MDMs) from 110 female goats were cultured and challenged in vitro with B. melitensis 16 M. After initial screening, 18 donor goats were selected based on their macrophages ability to restrict or allow bacterial intracellular growth and some elements of humoral and cellular immunity were studied in depth. MDMs that were able to restrict the pathogen's intracellular growth showed enhanced bacterial internalization, although there were no differences between groups in the production of reactive oxygen and nitrogen intermediates following 48 h treatment with heat-killed B. melitensis. Moreover, there were no differences between groups in the level of antibodies reacting with keyhole limpet hemocyanin (natural antibodies, NAbs) or with Brucella LPS antigens (cross-reacting antibodies, CrAbs), although a strong positive correlation between individual levels of IgM NAbs and IgM CrAbs was detected. Altogether, these results represent an initial step in understanding innate primary host response to B. melitensis, and deciphering which mechanisms may determine a successful outcome of the infection in goats.

Importance of brucellosis control programs of livestock on the improvement of one health.

Brucellosis not only represents an important health restraint on livestock but also causes high economic losses in many developing countries worldwide. Despite considerable efforts made for the control of brucellosis, the disease is still spreading in many regions (such as the Middle East) where it represents one of the most important health hazards impacting both animals and humans. The present review aims to investigate the efficacy of veterinary control programs regarding brucellosis, with a special focus on current prevention, control, and eradication approaches. The reasons for unsuccessful control programs such as the absence of highly effective vaccines and non-certified bulls are also debated, to understand why the prevalence of brucellosis in livestock is not decreasing in many areas despite considerable efforts taken to date. The importance of governmental and regional investment in brucellosis control remains one of the main limiting factors owing to the limited budget allocated to tackle this disease. In this context, one health concept has generated novel comprehensive approaches with multiple economic implications across the livestock industry and public health. However, the implementation of such global preventive strategies appears to be a key issue for many endemic and low-income countries. According to the collected data, epidemiological contexts including management and trade systems along with well-defined agro-ecological zones should be evaluated in brucellosis endemic countries to improve milk production and to enhance the sustainability of the livestock sector at both national and regional levels.

Seroprevalence of and Risk Factors Associated with Exposure to Brucella Spp. in Dairy Cattle in Three Different Agroecological Zones in Rwanda.

Livestock production is a key element for poverty alleviation, food security, and economic growth in Rwanda. In 2017, the national average milk production per cow was about 2.5 L per day; in 2020-2021, it is projected to increase to 3.5 L per day if improvement interventions including those designed to reduce the burden of brucellosis in cattle are implemented. The objective of the study reported here was to estimate the seroprevalence of and identify risk factors associated with dairy farms and cattle classified as seropositive to Brucella spp. in three different agroecological zones in Rwanda. Most study farms (40/85 or 47%) had one head of cattle only. Using the Rose Bengal test, the seroprevalence of brucellosis was 28/85 or 33% (95% CI = 24%, 43%) at the farm level and 63/465 or 14% (95% CI = 11%, 17%) at the animal level. Using logistic regression, at the farm level, the presence of seropositive cattle was associated with herd size (2-45 cattle, odds ratio = 21.2; 95% CI = 2.4, 184.5) (46-220 cattle, OR = 288.5; 95% CI = 24.3, 3,423.1) compared to farms with one animal, after controlling for main breed (local breeds, crossbreeds) on the farm. In addition, the odds of testing seropositive were 10.7 (95% CI = 2.3, 49.1) and 149.5 (95% CI = 19.3, 1,158.7) times higher in farms in Nyabihu district and Nyagatare district, respectively, than in farms in Muhanga district, after controlling for main breed on the farm. The odds of seropositivity to Brucella spp. were 2.8 times higher in farms with mostly local breeds, than in those with mostly crossbreeds; but the association was confounded by herd size and geographic location. At the animal level, the odds of seropositivity to Brucella spp. were 2.6 times higher in adult cattle than in young cattle (95% CI = 1.1, 6.3). Finally, we observed a high frequency of adult cattle (86%) and a high seroprevalence of brucellosis in adult cattle (25%) in Nyagatare; an indication that, in the absence of culling and other control measures, Brucella spp. infection pressure can be relatively constant and a steady source of disease transmission in pastoral systems in that district.

Comparative study of sodium bicarbonate- and magnesium hydroxide-based gastric antacids for the effectiveness of Salmonella delivered Brucella antigens against wild type challenge in BALB/c mice.

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.

Dexamethasone-induced stress exacerbates shedding, tissue antigen distribution, and pathology of caprine Brucellosis.

This study investigated dexamethasone-treatment, shedding routes, tissue antigen distribution, and pathology of caprine Brucellosis. Eighteen non-pregnant goats were randomly grouped into A, B, and C. Group A was administered dexamethasone for 7 days at 2 mg/kg before inoculating 0.5 mL B. melitensis at 107 CFU ocularly while group B was inoculated 0.5 mL B. melitensis only, and C as control negative. Blood samples, ocular, nasal, and vaginal swabs were obtained for evaluation. Three goats were sacrificed from each group at days 21 and 42 post-inoculation (pi) and selected tissues collected for PCR, histopathology, and immunohistochemistry. Brucella melitensis was detected in the ocular swabs of group A significantly higher than group B. Shedding was prolonged in group A compared to B. The overall shedding was 22.2% in group A and 9.4% in group B. The uterus of both groups A and B revealed mild inflammation and microgranuloma, extensive necrotic lesions in lymph nodes. Liver showed multifocal necrosis predominantly in group A. Lesion scoring showed significantly higher scores in A compared to B. Strong immunostaining was observed in the liver, lungs, and spleen, predominantly at day 21 pi. This study demonstrated dexamethasone prolonged shedding, tissue antigen distribution, and pathology in dexamethasone-treated goats.

Study on immunogenicity and antigenicity of a novel brucella multiepitope recombined protein.

Currently, brucellosis is a reemerged zoonotic infectious disease with an increased incidence in recent years. A simple, rapid and sensitive method for diagnosing brucellosis can help to reduce medical burden and economic loss. Previously, a multiple epitope recombinant protein was constructed based on linear B-cell epitope prediction tools. In this study, the recombinant protein was used as an antigen to study the immune response produced by immunized mice, and goat serum was used to verify its diagnostic accuracy. The production of antibodies was successfully induced in the vaccinated mice. Flow cytometric analysis revealed that the percentage of CD4+, CD8+ and the CD4+/CD8+ ratios were increased by T cell subsets in mouse splenocytes, indicating that the recombinant protein induced a strong immune response had strong immunoreactivity. Using indirect ELISA, the recombinant protein correctly diagnosed positive and negative brucellosis samples. Compared with the whole bacterial antigen, the recombinant protein had a weaker sensitivity but a stronger specificity. Animal experiments showed that the recombinant protein had good antigenicity, and indirect ELISA indicates that it can be used as an antigen to diagnose brucellosis. Therefore, the recombinant protein is a potential candidate antigen for brucellosis vaccine development and serological diagnosis.