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Bovine brucellosis (BB) is a cosmopolitan zoonotic disease caused by certain bacteria of the genus Brucella. It has negative health implications for the cattle, economic implications for the owners and is present in numerous cattle and wildlife populations worldwide. To determine the seroprevalence of BB in dual-purpose herds and assess its impact in the southern part of the Department of Atlántico, northern Colombia. 1,000 female cattle were tested for brucellosis, using the Rose Bengal Plate Test (RBPT) and indirect ELISA (iELISA) as screening and confirmatory tests, respectively. Of these, 100 animals tested positive by RBPT and 204 were inconclusive, while 38 positive animals were identified by iELISA. Consequently, the apparent seroprevalence was 3.8%, and the estimated true prevalence was 4.4%. Based on estimates of the economic losses caused by BB, the cost was calculated at $2,496,364 COP ($822 USD) per animal. The costs identified in this study include medication costs, loss of lactation, and others. It highlights the significant impact of BB on food security, particularly concerning the availability of and access to bovine food products, as well as on the economy of livestock farmers in the region. [ABSTRACT FROM AUTHOR]

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt. [ABSTRACT FROM AUTHOR]

Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression. [ABSTRACT FROM AUTHOR]

Brucellosis is a neglected zoonotic disease with significant public health impacts. It is primarily associated with classic Brucella species in mammals, but recent research has shown atypical Brucella strains in various hosts, including reptiles. Despite the known physiological differences between reptiles and humans, the rise in invasive reptile populations and other factors make reptiles and the pathogens they carry a potential public health concern. This study collected 90 venomous snakes from different regions in Iran and examined their liver samples, buccal, and cloaca cavities for Brucella spp. The samples underwent bacteriological analysis and DNA extraction. PCR amplification of the Omp28 gene and IS711-based PCR were used for the molecular identification of Brucella species. Subsequently, the Omp28 gene sequences were determined, and a phylogenetic tree was created. Brucella abortus was detected in the liver tissue of two snake species, namely Pseudocerastes persicus and Vipera albicornuta, at a prevalence rate of 2.2%. This finding is exceptional, as it has not been previously documented in venomous snake populations in the wild. The study confirms the presence of Brucella spp. in venomous snakes, suggesting the potential for zoonotic transmission. This discovery raises questions about the evolutionary forces shaping Brucella populations in reptiles. Understanding the prevalence and impact of Brucella in snakes is essential for wildlife conservation and assessing zoonotic risks. Proper safety measures are crucial when handling reptiles that may carry Brucella. Further research is needed to fill knowledge gaps regarding Brucella in these snake species and its potential zoonotic implications. [ABSTRACT FROM AUTHOR]

Brucellosis is regarded as one of the world's most severe zoonotic diseases. This study aimed to investigate the possibility of using recombinant Lactococcus lactis (L. lactis) as a live vector to produce recombinant Brucella abortus (B. abortus) Omp10. The gene sequences were obtained from GenBank. The proteins' immunogenicity was assessed using Vaxijen. After confirming the cloning of the Omp10 gene in the pNZ8148 vector by enzymatic digestion and PCR, transformation into L. lactis was done. SDS-PAGE and western blot methods evaluated omp10 protein expression. Mice received oral recombinant L. lactis vaccines. IgG antibodies against Omp10 were tested using ELISA. Real-time PCR and ELISA were used to analyze cytokine responses. Survival rate and histopathological changes were evaluated after the challenge. Omp10 was chosen for its 1.5524 antigenicity score. Enzymatic digestion and PCR identified a 381-bp gene fragment. A 10 kDa band indicated the success of L. lactis transformation. Mice administered the L. lactis-pNZ8148-Omp10-Usp45 vaccination 14 days after priming showed significantly higher Omp10-specific total IgG and IgG1 (P < 0.001) than the PBS control group. The mice who received the L. lactis-pNZ8148-Omp10-Usp45 and IRBA vaccines had significantly elevated levels of IFN-γ, TNFα, IL-4, and IL-10 in samples collected on days 14 and 28 (P < 0.001). Inflammatory response, morphological damage, alveolar edema, and lymphocyte infiltration were reduced in the target group. A recombinant L. lactis expressing the Omp10 protein was constructed as an oral Lactococcus-based vaccine and compared to live attenuated vaccines for future brucellosis investigations. [ABSTRACT FROM AUTHOR]

A proof of principle biosensor for the Brucella abortus recognition onsite is presented. The system is based on a plasmonic optical fiber probe functionalized with an oriented antibody layer immobilized on a short polyethyleneglycol (PEG) interface through carbodiimide chemistry and protein G as an intermediate layer. The biosensor is inserted in a holder built in 3D printing technology, obtaining a custom holder useful for housing the sample to be measured and the equipment. The removable sensor chip is a low-cost Surface Plasmon Resonance (SPR) platform based on D-shaped plastic optical fibers (POFs), built-in in 3D printed connectors, used here for the first time to detect bacteria via a bio-receptor layer specific for its membrane protein. The performances of the biosensor in Brucella abortus recognition are tested by using two different SPR-POF probes combined with the same bio-receptor layer. The best sensor configuration has presented a sensitivity at low concentrations of one order of magnitude greater than the other. A limit of detection (LoD) of 2.8 bacteria/mL is achieved well competitive with other systems but without the need for amplification or special sample treatments. Specificity has been tested using Salmonella bacteria, and reproducibility, regenerability and stability are moreover evaluated. These experimental results pave the way for building an efficient and specific biosensor system for Brucella abortus detection onsite and in a few minutes. Moreover, the proposed POF-based SPR biosensor device, with respect to the already available technologies, could be a Point-of-care-test (POCT), simple to use, small-size and portable, low-cost, don't necessary of a microfluidic system, and can be connected to the Internet (IoT). [ABSTRACT FROM AUTHOR]

Brucellosis is a chronic zoonotic disease caused by brucella strains that adversely affects human and animal health. Increases or decreases in acute phase protein (APP) levels are observed in various infective diseases in cattle. In our study, it was aimed to investigate how the change in APP levels in cattle infected with Brucella abortus and which ones can be used as a marker. In the study, blood serum and plasma of 28 brucella and 10 healthy cattle collected from various cattle farm in Samsun were used. Rose bengal plate test and complement fixation test were applied to the serums. While serum amyloid A (SAA), haptoglobin (Hp), albumin, and total protein (TP) levels were determined in the blood serums taken from the groups, fibrinogen (Fb) level was determined in the plasma. As a result of the analysis, SAA 350.2 mg/L, Hp 1.57 g/L, Fb 14.58 µg/mL, TP 8.99 g/L, and albumin 2.99 g/L were found in the group infected with B. abortus. SAA 81 mg/L, Hp 0.41 g/L, Fb 2.1 µg/mL, TP 6.21 g/L, and albumin 3.57 g/L were found in the control group. Comparing the APP levels of cattle infected with B. abortus and the cattle in the control group, the levels of SAA (P < 0.05), Hp (P < 0.05), Fb (P < 0.05), and Tp (P < 0.05) were statistically significantly higher in the infected group, while the level of albumin (P < 0.05) was found to be statistically significantly decreased. [ABSTRACT FROM AUTHOR]

It is now clearly recognized that light modulates the physiology of many bacterial chemotrophs, either directly or indirectly. An interesting case are bacterial pathogens of clinical relevance. This work summarizes, discusses, and provides novel complementary information to what is currently known about light sensing and responses in critical human pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus. These pathogens are associated with severe hospital and community infections difficult to treat due to resistance to multiple drugs. Moreover, light responses in Brucella abortus, an important animal and human pathogen, are also compiled. Evidence recovered so far indicates that light modulates aspects related to pathogenesis, persistence, and antibiotic susceptibility in these pathogens; such as motility, biofilm formation, iron uptake, tolerance to antibiotics, hemolysis and virulence. The pathogens elicit differential responses to light depending likely on their pathophysiology, ability to cause disease and characteristics of the host. The response to light is not restricted to discrete physiological traits but is global. In higher organisms, light provides spatial and temporal information. Then, it is crucial to understand what information light is providing in these bacterial pathogens. Our current hypothesis postulates that light serves as a signal that allows these pathogens to synchronize their behavior to the circadian rhythm of the host, to optimize infection. Advances on the molecular mechanism of light signal transduction and physiological responses to light, as well as in the relation between light and bacterial infection, would not only enlarge our understanding of bacterial pathogenesis but also could potentially provide alternative treatment options for infectious illnesses. [ABSTRACT FROM AUTHOR]

Brucella melitensis primarily affects sheep, goats and is associated with brucellosis in humans, which is one of the world's most widespread neglected zoonotic disease. The current study attempted the determination of genetic diversity through comparative genome analysis of B. melitensis strains reported from India with other countries. The study also reports the isolation and identification of B. melitensis BMNDDB8664 from a cow with a history of abortion, whole-genome sequencing (WGS), determination of virulence factors, genotyping, and comparative genome analysis. Multilocus sequence typing, Multiple locus variable number of tandem repeats analysis (MLVA), and WGS based phylogeny revealed the predominance of ST-8 and genotypes (116 and II respectively) that clustered to the East Mediterranean lineage. Identification of hitherto unreported genotypes by MLVA also indicated the existence and circulation of West Mediterranean and American lineages in India. Though the AMOS-PCR results suggest the BMNDDB8664 isolate as Brucella abortus, the outcomes from multiplex PCR, ribosomal multilocus sequence typing, and WGS analysis confirmed it as B. melitensis. The analysis revealed the presence of adeF gene (aids conferring resistance to fluoro-quinolone and tetracyclines). The isolate lacked two important T4SS genes virB2 and virB7 genes (roles in infection and rifampicin resistance respectively) and also lacked the Brucella suis mprF gene that aids intracellular survival. Further, BMNDDB8664 lacked some of the genes associated with LPS synthesis (wbkB, wbkC) and transport (wzm, wzt) and hence, is most likely a rough strain. WGS-based phylogenetic analysis revealed close genetic relatedness of this BMNDDB8664 with a sheep isolate and two human isolates. The results prompt systematic, broad-based epidemiological studies on brucella infection at the species level. For effective control of human brucellosis, a concerted One Health approach with studies encircling the identification of aetiology at species, strain level to find their prevalence, spread, and inter-host transmission patterns need to be understood, for better design and implementation of effective control strategies in India and other endemic regions. [ABSTRACT FROM AUTHOR]

The development of multifunctional particles using polymeric scaffolds is an emerging technology for many nanobiotechnological applications. Here we present a system for the production of multifunctional complexes, based on the high affinity non-covalent interaction of cohesin and dockerin modules complementary fused to decameric Brucella abortus lumazine synthase (BLS) subunits, and selected target proteins, respectively. The cohesin-BLS scaffold was solubly expressed in high yield in Escherichia coli, and revealed a high thermostability. The production of multienzymatic particles using this system was evaluated using the catalytic domain of Cellulomonas fimi endoglucanase CenA recombinantly fused to a dockerin module. Coupling of the enzyme to the scaffold was highly efficient and occurred with the expected stoichiometry. The decavalent enzymatic complexes obtained showed higher cellulolytic activity and association to the substrate compared to equivalent amounts of the free enzyme. This phenomenon was dependent on the multiplicity and proximity of the enzymes coupled to the scaffold, and was attributed to an avidity effect in the polyvalent enzyme interaction with the substrate. Our results highlight the usefulness of the scaffold presented in this work for the development of multifunctional particles, and the improvement of lignocellulose degradation among other applications. Key points: • New system for multifunctional particle production using the BLS scaffold • Higher cellulolytic activity of polyvalent endoglucanase compared to the free enzyme • Amount of enzyme associated to cellulose is higher for the polyvalent endoglucanase [ABSTRACT FROM AUTHOR]

Bovine brucellosis is a disease that significantly impacts animal production and human health. Although many sensitive diagnostic tests are used, there is still no ideal fast serological test for all epidemiological situations. In this context, we developed peptides that mimic regions of antigenic proteins of Brucella abortus and can be used in serological diagnosis. Results: From phage display technology, we randomly selected nine clones of phage displaying peptide binders to B. abortus. These clones were sequenced and translated. After molecular docking analysis, two peptides (Ba4 and Ba9) were selected, chemically synthesized, and verified for their potential diagnostic value. By enzyme-linked immunoassay (ELISA), Ba9 showed a sensitivity of up to 97.5% to detect antibodies circulating in animals with brucellosis. We incorporated the peptide Ba9 onto a bioelectrode (graphite modified with poly-3-hydroxyphenylacetic acid). Then, direct serum detection was demonstrated by differential pulse voltammetry, micrographs, and topographic analyses in addition to the average roughness coefficient (Ra) and the value of the mean squared deviation of the roughness (Rms). Conclusion: This work shows that the mimetic epitope of B. abortus can be useful for developing new platforms for diagnosing brucellosis. In addition, we propose a fast test based on an electrochemical sensor using graphite modified with poly-3-hydroxyphenylacetic acid. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide is essential for most Gram-negative bacteria as it is a main component of the outer membrane. In the pathogen Brucella abortus, smooth lipopolysaccharide containing the O-antigen is required for virulence. Being part of the Rhizobiales, Brucella spp. display unipolar growth and lipopolysaccharide was shown to be incorporated at the active growth sites, i.e. the new pole and the division site. By localizing proteins involved in the lipopolysaccharide transport across the cell envelope, from the inner to the outer membrane, we show that the lipopolysaccharide incorporation sites are determined by the inner membrane complex of the lipopolysaccharide transport system. Moreover, we identify the main O-antigen ligase of Brucella spp. involved in smooth lipopolysaccharide synthesis. Altogether, our data highlight a layer of spatiotemporal organization of the lipopolysaccharide biosynthesis pathway and identify an original class of bifunctional O-antigen ligases. Pathogenic Brucella abortus, containing a mix of lipopolysaccharides with or without O-antigen, grows its envelope in a unipolar manner. Here, Servais et al, localize the LPS translocation machinery and identify the main O-antigen ligase in Brucella species, shedding light on the basic biology of this organism. [ABSTRACT FROM AUTHOR]

The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. Here we identify two Brucella abortus effectors, NyxA and NyxB, that interfere with host protease SENP3, and this facilitates intracellular replication of the pathogen. The translocated Nyx effectors directly interact with SENP3 via a defined acidic patch (identified from the crystal structure of NyxB), preventing nucleolar localisation of SENP3 at late stages of infection. By sequestering SENP3, the effectors promote cytoplasmic accumulation of nucleolar AAA-ATPase NVL and ribosomal protein L5 (RPL5) in effector-enriched structures in the vicinity of replicating bacteria. The shuttling of ribosomal biogenesis-associated nucleolar proteins is inhibited by SENP3 and requires the autophagy-initiation protein Beclin1 and the SUMO-E3 ligase PIAS3. Our results highlight a nucleomodulatory function of two Brucella effectors and reveal that SENP3 is a crucial regulator of the subcellular localisation of nucleolar proteins during Brucella infection, promoting intracellular replication of the pathogen. The bacterium Brucella abortus is an intracellular pathogen that modulates autophagy in host cells. Here, the authors identify two B. abortus effectors that interact with host protease SENP3, thus promoting cytoplasmic accumulation of nucleolar proteins associated with ribosomal biogenesis and facilitating intracellular replication of the pathogen [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) are a group of functional RNA molecules without protein-coding potential and play vital roles in majority of biological processes. To date, the expression profiles of lncRNAs and their influence on Brucella replication in RAW264.7 cells are poorly understood. In this study, we performed high-throughput transcriptome analysis to investigate the differentially expressed lncRNAs associated with Brucella abortus S2308 infection. Of these, 8, 6, 130 and 94 cellular lncRNAs were differentially expressed at 4, 8, 24 and 48 h post-infection, respectively. Moreover, 1918 protein-coding genes are predicted as potential cis target genes of differentially expressed lncRNAs by searching protein-coding genes located at upstream and downstream of lncRNA loci on the chromosome DNA of Mus musculus. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that majority of lncRNA target genes were associated with B. abortus infection. Fourteen lncRNAs from transcriptome data were selected for qRT-PCR verification, confirming 13 were differentially expressed. Animal experiments revealed three were differentially expressed in vivo by qRT-PCR analysis. Furthermore, knockdown of LNC_000428 by CRISPR/dCas9 inhibition or Locked Nucleic Acids transfection downregulated Tnfrsf8 expression at mRNA level and increased Brucella intracellular replication. Thus, we provide a novel evidence that lncRNAs induced by Brucella-infection function on Brucella intracellular replication. [ABSTRACT FROM AUTHOR]

To effectively cut off the transmission of Brucella to humans through milk, a novel biosensor for detection of Brucella was constructed using cysteamine functionalized nanogold (Cys-Au) and 4-Mercaptobenzoic acid (4-MBA) modified gold electrode as the platform. The morphology of the prepared nanomaterials was characterized by laser particle sizer, XPS and SEM, and the results showed the 4-MBA film was modified on the gold electrode surface, and the diameter of successfully prepared nanogold and Cys-Au are about 55 and 65 nm, respectively. The electrochemical performance of the biosensor was tested in Brucella antigen solution by square wave voltammetry (SWV). The results showed the reproducibility and stability of the biosensor were good, the spiked recovery in milk was in the range of 95.12–105.27%, and the minimum detection limit (S/N = 3) was 5.12 × 102 cfu/mL with a linear range from 1.6 × 102 to 1.6 × 108 cfu/mL. In addition, the minimum detection limit was reduced from 5.12 × 102 cfu/mL to 7.0 × 101 cfu/mL after processing the SWV data by second-order derivative transformation, which showed Second-order derivative transformation is very helpful to further optimize the performance of the sensor. Compared with the previously reported test methods, the prepared biosensors have a wider linear range and lower limit of detection (LOD). Therefore, the constructed biosensor can be used as a promising tool for the trace detection of Brucella in milk samples. [ABSTRACT FROM AUTHOR]

The receptor binding domain (RBD) of the Spike protein from SARS-CoV-2 is a promising candidate to develop effective COVID-19 vaccines since it can induce potent neutralizing antibodies. We have previously reported the highly efficient production of RBD in Pichia pastoris, which is structurally similar to the same protein produced in mammalian HEK-293T cells. In this work we designed an RBD multimer with the purpose of increasing its immunogenicity. We produced multimeric particles by a transpeptidation reaction between RBD expressed in P. pastoris and Lumazine Synthase from Brucella abortus (BLS), which is a highly immunogenic and very stable decameric 170 kDa protein. Such particles were used to vaccinate mice with two doses 30 days apart. When the particles ratio of RBD to BLS units was high (6–7 RBD molecules per BLS decamer in average), the humoral immune response was significantly higher than that elicited by RBD alone or by RBD-BLS particles with a lower RBD to BLS ratio (1–2 RBD molecules per BLS decamer). Remarkably, multimeric particles with a high number of RBD copies elicited a high titer of neutralizing IgGs. These results indicate that multimeric particles composed of RBD covalent coupled to BLS possess an advantageous architecture for antigen presentation to the immune system, and therefore enhancing RBD immunogenicity. Thus, multimeric RBD-BLS particles are promising candidates for a protein-based vaccine. [ABSTRACT FROM AUTHOR]

Seroprevalence and risk factors of bovine brucellosis (Brucella abortus) in herds and cattle were estimated by a cross-sectional study in the state of Paraná, Brazil. The state was divided into seven regions and a random, two-stage sampling was performed on properties and cattle from each region between 2018 and 2019. Serum samples were collected from 11,592 cows over 24 months from 1,757 properties and a questionnaire was applied to identify potential risk factors. As recommended by the National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), serological testing for the detection of anti-Brucella antibodies included the buffered plate agglutination test (screening test) and the fluorescence polarization assay (confirmatory test). The seroprevalence of bovine brucellosis on properties and in cattle was 4.87% (95% confidence interval [CI]: 3.98–5.93%) and 2.24% (95% CI: 1.47–3.41%), respectively. Multiple logistic regression analysis identified larger herd size and failure to test for brucellosis as risk factors for the presence of anti-B. abortus antibodies. These results demonstrate no change in the prevalence when comparing initial studies conducted in 2002. Given our findings, it is recommended that policies for brucellosis control include a widespread vaccination program for higher prevalence areas and eradication approach to lower prevalence areas. All steps related to correct immunization of the herds should be verified and improved by training and education. Health education action must be carried out informing farmers about the risks of introducing animals not tested for brucellosis into their herds and the benefits of testing their herds regularly. [ABSTRACT FROM AUTHOR]

Brucellosis is considered as the most common bacterial zoonosis in the world. Although the laboratory findings are the most reliable diagnosis today, the current laboratory methods have many limitations. This research aimed to design and evaluate the performance of a novel technique based on the localized surface plasmon resonance (LSPR) to eliminate or reduce existing shortcomings. For this purpose, smooth lipopolysaccharides were extracted from Brucella melitensis and Brucella abortus and fixed on the surface of the gold nanoparticles through covalent interactions. After some optimizing processes, dynamic light scattering was used to characterize the probe. The detection of captured anti-Brucella antibody was performed by measuring the redshift on LSPR peak followed by the determination of cutoff value, which indicated a significant difference between controls and true positive patients (P value < 0.01). Furthermore, 40 sera from true negative samples and positive patients were used to evaluate the performance of this method by comparing its outcomes with the gold standard (culture), standard tube agglutination test, and anti-brucellosis IgM and IgG levels (ELISA). The sensitivity, specificity, positive predictive value, and negative predictive value showed an appropriate performance of the LSPR-based method (85%, 100%, 100%, and 86%, respectively). The current research results provide a promising fast, convenient, and inexpensive method for detecting the anti-Brucella antibodies in human sera, which can be widely used in medical laboratories to diagnose brucellosis quickly and effectively. [ABSTRACT FROM AUTHOR]

Heat shock proteins are molecular chaperones that are immunogens as well as potent inducers of an antigen-specific immunological response. In this study, we aimed to evaluate if co-immunization of Brucella rOmp22 and rDnaK proteins had boosted immunogenic activity as compared to rOmp22 immunization alone in mice. For this, gene-encoding DnaK of B. abortus was cloned, expressed in E. coli and purified using Ni–NTA agarose. Immuno-modulatory effect of rDnaK protein was evaluated in mice when co-immunized with Brucella rOmp22. Four groups of mice (n = 6 per group) were used in the study. The control group was immunized with rOmp22 alone, while rOmp22 emulsified with conventional adjuvants (Freund's complete and incomplete adjuvants) and rOmp22 mixed with rDnaK were injected to group I and group II in mice, respectively. Group III mice were immunized with rDnaK alone. IgG class switching (IgG1 and IgG2a) response to immunization was assessed by enzyme-linked immunosorbent assay and expression of IL-4 and IL-12 mRNA was assessed by real-time PCR to evaluate the immune response in mice. The ratio of IgG1–IgG2a was less than 1 in mice co-immunized with rOmp22 and rDnaK, indicating that the immune response was directed towards CMI arm in this group of mice. Moreover, IL-12 mRNA expression was also up-regulated to a greater extent in mice co-immunized with rOmp22 and rDnaK as compared to those immunized with rOmp22 along with the conventional adjuvants, or rOmp22 alone. Our data suggest that rDnaK could be responsible for modulating the immune response, specifically the CMI response. [ABSTRACT FROM AUTHOR]

This study analyzed the difference between biofilm and planktonic Brucella abortus using metabolomics and proteomics. Brucella abortus was cultured in different media to induce Brucella abortus biofilm formation and planktonic cells, followed by metabolomics and proteomics analyses for these two samples. Significant differential metabolites were identified, followed by KEGG pathway analysis. Differentially expressed proteins were identified, followed by subcellular localization, GO annotation, and KEGG pathway enrichment. Additionally, a correlation analysis of metabolomics and proteomics was performed. Metabolomics analysis showed 7682 positive and 4433 negative metabolites, including 188 positive and 117 negative significant differential metabolites. These differential metabolites were enriched in fatty acid/unsaturated fatty acid biosynthesis and linoleic acid metabolism. Proteomics analysis revealed 1759 proteins, including 486 differentially expressed proteins, which were enriched in various metabolic and degradation-related pathways. Subcellular localization showed that 74.3% of the differential proteins were cytoplasmic proteins. Correlation analysis showed that 1-palmitoyl-2-oleoyl-phosphatidylglycerol had the most significant correlations with proteins, followed by cytosine. Both metabolites correlated with the protein Q57EI7 (RbsB-1, ribose ABC transporter). One common pathway, fatty acid biosynthesis, was identified by both proteomics and metabolomics analyses that involved the metabolites, oleic acid, and protein Q57DK3 (biotin carboxylase). There were metabolomic and proteomic differences between Brucella abortus biofilm and planktonic cells, and these results provide novel insights into the biofilm-forming process of Brucella abortus. [ABSTRACT FROM AUTHOR]

The field of landscape genetics enables the study of infectious disease dynamics by connecting the landscape features with evolutionary changes. Quantifying genetic correlation across space is helpful in providing insight into the rate of spread of an infectious disease. We investigate two genetic patterns in spatially referenced single-nucleotide polymorphisms (SNPs): isolation by distance and isolation by resistance. We model the data using a Generalized Linear Mixed effect Model (GLMM) with spatially referenced random effects and provide a novel approach for estimating parameters in spatial GLMMs. In this approach, we use the links between binary probit models and bivariate normal probabilities to directly compute the model-based covariance function for spatial binary data. Parameter estimation is based on minimizing sum of squared distance between the elements of sample covariance and model-based covariance matrices. We analyze data including Brucella Abortus SNPs from spatially referenced hosts in the Greater Yellowstone Ecosystem. [ABSTRACT FROM AUTHOR]

Brucellosis is among the most important zoonotic infectious diseases worldwide affecting both humans and domestic animals. The present study aimed to determine and compare the seroprevalence of brucellosis among rural and periurban dairy cattle farms of four Iranian provinces from 2017 to 2019. We applied different serological tests, including RBT, SAT, and iELISA to evaluate the brucellosis prevalence among 2808 dairy cattle. Species-specific multiplex PCR and biotyping tests were also used to further identify the implicated Brucella species. Serological screening using RBT, SAT, and iELISA led to 157 (5.6%), 112 (3.9%), and 139 (4.9%) positive results among tested cattle, respectively. Brucella abortus biovars 1 (2 cases) and biovars 3 (42 cases) were identified by biotyping experiments and multiplex PCR in all 44 tested lymph node samples. Further, Cohen’s kappa statistical analysis revealed that the best degree of agreement was seen between RBT and iELISA (99.4%), followed by SAT/iELISA (98.5%) and finally RBT/SAT (98.4%). Our results also showed a significantly lower seroprevalence of brucellosis in periurban dairy cattle when compared to rural dairy cattle population (p value= 0.01). These results reflect the need for better vaccine coverage using RB51 combined with an appropriate test-and-slaughter program in the rural dairy cattle population. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the possible alterations in some cytokines and oxidative stress biomarkers in cows with acute Brucella abortus (B. abortus) infection. The study has been carried out on twenty-two cows' sera. All serum samples were examined for brucellosis by indirect enzyme-linked immunosorbant assay (i-ELISA). Cows that were seronegative for B. abortus infection were used as a control group; meanwhile, cows that were diagnosed positive for B. abortus infection were used as an infected group. Cytokine profile and oxidative state markers showed significant increases in interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), and nitric oxide (NO) levels in B. abortus-infected cows in comparison with the control group. On the other hand, significant decreases in interleukin-10 (IL-10) and total antioxidant capacity (TAC) levels were reported in B. abortus-infected cows when compared with controls. [ABSTRACT FROM AUTHOR]

Brucellosis is a zoonosis caused by bacteria of the Brucella genus. Any source of contamination that could be infectious must be monitored to reduce the risk of exposure to brucellosis, so the purpose of this work was to determine the presence of Brucella spp. on surface water and tilapia (Oreochromis niloticus) skin from a volcanic lake in Mexico. A seasonal sampling during 2016–2017 was carried out at fifteen specific sites for water sampling and five sites for the collection of tilapia fish. From all water and fish samples tested, we found only three isolates of Brucella species. We isolated and identified B. abortus from surface water through bacteriological and molecular techniques, and B. abortus and B. suis from the same tilapia skin sample. The isolated strains likely came from breeding animals that are common to the region, such as infected pigs or cattle with Brucella abortus or B. suis, respectively. A similar finding has not been reported in a water from volcanic lake or tilapia fish in Mexico. We concluded that B. abortus and B. suis are present on the surface water of the volcanic lake and tilapia skin as possible contaminants derived from biological material from cows and pigs carrying this bacterium. [ABSTRACT FROM AUTHOR]

Current scenario in communicable diseases has generated new era that identifies the "One health" approach to understand the sharing and management of etiological agents with its impact on ecosystem. Under this context the relevance of zoonotic diseases generates major concern. The indiscriminate and higher use of antibiotics in animal husbandry creates substantial pressure on the gut microbiome for development of resistance due to shorter generation time and high density. Thus, gut works as a bioreactor for the breeding of ARBs in this scenario and are continuously released in different niches. These ARBs transfer resistance genes among native flora through horizontal gene transfer events, vectors and quorum sensing. About 60% of infectious diseases in human are caused by zoonotic pathogens have potential to carry ARGs which could be transmitted to humans. The well documented zoonotic diseases are anthrax cause by Bacillus anthracis, bovine tuberculosis by Mycobacterium tuberculosis, brucellosis by Brucella abortus, and hemorrhagic colitis by Escherichia coli. Similarly, most of the antibiotics are not completely metabolized and released in unmetabolized forms which enters the food chain and affect various ecological niches through bioaccumulation. The persistence period of antibiotics ranges from < 1 to 3466 days in environment. The consequences of misusing the antibiotic in livestock and their fate in various ecological niches have been discussed in this review. Further the light sheds on antibiotics persistence and it biodegradation through different abiotic and biotic approaches in environment. The knowledge on personnel hygiene and strong surveillance system for zoonotic disease including ARBs transmission, prevention and control measures should be established to regulate the spread of AMR in the environment and subsequently to the human being through a food web. [ABSTRACT FROM AUTHOR]

Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10–100 CFU) required to establish the infection. In this study, we developed sandwich ELISA with detection range of 102 to 108 cells mL−1 and limit of detection at 103 cells mL−1 by employing polyclonal rabbit IgG (capture antibody, 10 µg mL−1) and mice IgG (detection antibody, 50 µg mL−1) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 102 cells mL−1 along with non co-operative interaction of protein albumin. Further, kinetic evaluation study using detection antibody against cell envelope antigen was performed whereby, Equilibrium Dissociation Constant (KD) and Maximum Binding Capacity (Bmax) were found to be 16.48 pM and 81.67 m° for Brucella abortus S99 and 0.42 pM and 54.50 m° for Brucella melitensis 16 M, respectively. During interference study, sandwich ELISA assay cross-reacted with either of the polyclonal antibody of above Brucella species. Upon validation, no cross-reactivity observed with bacteria-closely related to Brucella. In conclusion, developed semi-quantitative sandwich immunoassay is sensitively rapid in whole cell detection of Brucella and will be useful in development of detection assays from environmental and clinical matrices. [ABSTRACT FROM AUTHOR]

A cross-sectional study was conducted to determine the seroprevalence of Brucella spp. and Leptospira spp. and risk factors of exposure in cattle in three zones with varying land use types and wildlife–livestock interactions. Five villages were selected purposively; two in areas with intensive livestock–wildlife interactions (zone 1), another two in areas with moderate livestock–wildlife interactions (zone 2) and one in areas where wildlife–livestock interactions are rarer (zone 3). Sera samples were collected from 1170 cattle belonging to 390 herds in all the zones and tested for antibodies against Brucella abortus and Leptospira interrogans serovar hardjo using ELISA kits. Data on putative risk factors for seropositivity of these pathogens in cattle were collected using a questionnaire. The overall apparent animal-level seroprevalence of brucellosis and leptospirosis was, respectively, 36.9% (95% CI 34.1–39.8) and 23.5% (95% CI 21.1–26.0). Brucella spp. seroprevalence was higher in zone 1 than in zones 2 and 3 (χ2 = 25.1, df = 2, P < 0.001). Zones 1 and 2 had significantly higher Leptospira spp. seroprevalence than zone 3 (χ2 = 7.0, df = 2, P = 0.029). Results of multivariable analyses identified animal sex (female) and zones (high interface area) as significant predictors (P < 0.05) of animal-level seropositivity of Brucella spp. For Leptospira spp., important predictors of animal-level seropositivity were animal sex (female), zones (moderate interface area) and herds utilizing a communal grazing reserve. The seroprevalences of Brucella spp. and Leptospira spp. in cattle were higher in areas with moderate to high wildlife–livestock interactions than those with rare interactions. [ABSTRACT FROM AUTHOR]

It is assumed that intracellular pathogenic bacteria have to cope with DNA alkylating stress within host cells. Here we use single-cell reporter systems to show that the pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophage infection. Genes encoding direct repair and base-excision repair pathways are required by B. abortus to face this stress in vitro and in a mouse infection model. Among these genes, ogt is found to be under the control of the conserved cell-cycle transcription factor GcrA. Our results highlight that the control of DNA repair in B. abortus displays distinct features that are not present in model organisms such as Escherichia coli. It is assumed that intracellular pathogenic bacteria must cope with DNA alkylating stress within host cells. Here, Poncin et al. show that the pathogen Brucella abortus does encounter alkylating stress within macrophages, and shed light into the pathways required for DNA repair in this organism. [ABSTRACT FROM AUTHOR]

Brucellosis is primarily a zoonotic disease caused by members of the Brucella genus which consists of 11 recognized species based on pathogenicity and host preferences. Peptide vaccines are an alternative strategy to conventional vaccines based on the use of short peptide sequences and engineered to induce highly targeted immune responses, avoiding allergenic and/or reactogenic sequences. In this study, antigenic peptide sequence of Brucella abortus, WLAEIKQRSLMVHG, was chemically synthesized by adding tryptophan to the N-terminus of sequence, purified and characterized for the first time in the literature. Molecular weight of the peptide was determined by using LC–MS. A linear response (R2 > 0.998) is observed for peptide in the range of 2–250 µg/mL. The LOD and LOQ values are 0.08 and 0.27 µg/kg respectively. Precision and accuracy ranges were found to be %RSD < 0.2 and 57.3–103.2%, respectively. Fluorescence property of the peptide was shown via fluorescence spectroscopy measurement. The 3D de novo structure of the peptide was predicted with PEP-FOLD server. According to the data obtained from the lipophilicity study (LogD7.4 = − 3.093 ± 0.195), the peptide can not cross the blood–brain barrier if applied via intravenously. This has shown us that peptide can be used as a peptide candidate at the vaccine studies. The production of antigenic peptides as in this study is the main component of the preparation of synthetic vaccine systems. [ABSTRACT FROM AUTHOR]

We studied the expression of activation markers CD25 and CD69 by blood lymphocytes in white mice vaccinated with Brucella abortus 19 BA in antigen-specific tests in vitro. During incubation of blood lymphocytes with brucellosis polysaccharide-protein antigen, a statistically significant increase in the expression of CD25 by B cells and CD69 by T cells was observed; brucellin increased the expression of CD25 by B and T cells. Comparative analysis of the action of antigen preparations B. abortus showed that only brucellin has antigen-specific activity against CD19+CD25+ cells. The used method can be considered as a promising test for evaluation of the effectiveness of brucellosis immunoprophylaxis, which substantiates the need for further research. [ABSTRACT FROM AUTHOR]

The objective of this survey was to assess the prevalence of brucellosis with focus on Brucella abortus and Brucella melletensis in different kinds of cats in north-east of Iran. Different kinds of cats included stray, pet, and cats which were located in dairy cattle herds tested. Blood sera of 48 (34.28%) stray, 42 (30%) pet, and 50 (35.71%) cats collected from 15 industrial dairy cattle herds. They were used for rapid and tube agglutination tests such as Rose Bengal, Wright, and 2-mercaptoethanol (2-ME). The comparison among age, sex, weight, and lifestyle of cats with serological tests of brucellosis were examined statistically. Totally, eight (5.71%) cats were mild positive with low titer. Only two (1.42%) cats reacted in 2-ME. There was no statistically significant relationship between sex of cat (male vs female) and serological results (P = 0.07). However, comparison among different age groups revealed significant differences (p = 0.009). There were no significant differences between the infected cats collected from dairy cattle herds and stray cats, but the stray cats and pet cats showed the significant differences (p = 0.028). This survey showed that Brucella infection (1) mainly occurred in male and stray cats aged 1-2 years and (2) is transiently mild, and (3) negative brucellosis in pet cats represents optimal hygiene and health monitoring of these cats by the owners. So, stray cats can be a risk factor for brucellosis in human and animal populations in north-east of Iran; thus, strong precautions over stray cats are necessary. [ABSTRACT FROM AUTHOR]

Brucella poses a great threat to animal and human health. Vaccination is the most promising strategy in the effort to control Brucella abortus (B. abortus) infection, but the currently used live vaccines interfere with diagnostic tests and could potentially result in disease outbreak. Therefore, new subunit vaccines and combined immunization strategies are currently under investigation. In this study, immunogenicity and protection ability of a recombinant adenovirus and plasmid DNA vaccine co-expressing P39 and lumazine synthase proteins of B. abortus were evaluated based on the construction of the two molecular vaccines. Four immunization strategies (single adenovirus, single DNA, adenovirus/DNA, DNA/adenovirus) were investigated. The results showed that the immunization strategy of DNA priming followed by adenovirus boosting induced robust humoral and cellular immune responses, and it significantly reduced the numbers of B. abortus in a mouse model. These results suggest that it could be a potential antigen candidate for development of a new subunit vaccine against B. abortus infection. [ABSTRACT FROM AUTHOR]

Bovine brucellosis is endemic in Colombia, and is a mandatory notifiable disease, subjected to a control program based on four surveillance procedures: passive surveillance, test-and-remove, certification of disease-free farms, and animal movements. The objective of this study is to estimate the evolution of bovine brucellosis in Colombia over a 7-year period (2006-2012) using data from the official control program. A total of 58 epidemiologic variables were analyzed for each year at the department level. Univariate descriptive analysis and principal components analysis (PCA) were performed to ascertain the behavior of the variables. These programs covered 3% of the census in 2006, increasing to 15% in 2012. The percentage of positive farms averaged 22% in 2006 and 23% in 2012. The highest proportion of positive farms was in the Orinoquía region (24.6 to 49.6%); the lowest was in the Amazon region, (17.9 to 32.7%). The percentage of positive animals presented certain differences between years but without any clear trend (4.7% in 2006 and 4.6% in 2012), indicating that the brucellosis control program had a low impact in Colombia in these years. The results for each surveillance procedure were 6.8% for passive surveillance, 5.9% for test-and-remove, and 4.4% both in disease-free farms and in animal movement tests. The results obtained by PCA led to finding three different clusters: geographic areas with low bovine production and low bovine brucellosis surveillance, areas with medium bovine production and medium surveillance for bovine brucellosis, and areas with a predominant bovine production, applying sanitary measures to control bovine brucellosis. [ABSTRACT FROM AUTHOR]

This study aims to investigate the effects of Trypanosoma brucei infection and treatment on haematoserological status of West African Dwarf sheep to Brucella abortus vaccine. Twenty-four adult WAD sheep of mixed sexes were randomly assigned into 6 groups of 4 each. They were either infected with 1 × 10 trypanosomes intravenously (groups 2-6) or uninfected (group 1). The different groups were treated as follows: group 2 (positive control-7 mg/kg diminazene aceturate (DA)), group 3 (7 mg/kg DA and 5 mg/kg levamisole), group 4 (7 mg/kg DA and 50, 000 IU vitamin A), group 5 (7 mg/kg DA and 50, 000 IU vitamin A 3 weeks post infection), and group 6 (7 mg/kg DA and 5 mg/kg levamisole and 50, 000 IU vitamin A). All the animals were vaccinated with B. abortus S19 vaccine 2 weeks post infection (PI). Parameters assessed parasitaemia, total and differential white blood cell counts, and antibody titres to B. abortus antigen. Average prepatent period was 5 days. Trypanosomes were completely cleared from the bloodstream following treatment. There was no relapse parasitaemia recorded. By weeks one and two post infection, there was significant ( P < 0.05) leucocytosis which was due to lymphocytosis and neutrophilia in all infected groups. There were no significant changes in monocytes, oesinophils, basophils, and antibody titres of both infected and uninfected sheep. Experimental T. brucei infection and treatment of WAD sheep caused significant ( P < 0.05) leucocytosis 1 to 2 weeks PI due to lymphocytosis and neutrophilia, but no significant differences in antibody titres to B. abortus S19 vaccine when compared with the vaccinated uninfected. [ABSTRACT FROM AUTHOR]

Brucellosis in humans is one of the most important zoonotic diseases worldwide. This disease causes weakness, fever, pain, sweats, and complications in the liver, spleen, and lymphatic system; Brucellosis requires long-term antibiotic therapy. This study attempts to identify Brucella abortus via an immunofluorescence (IF) test considering antibody against an outer membrane protein with 16-kDa molecular weight (OMP16). The B. abortus OMP16 protein was synthesized through cloning and expressing of the related gene and finally was purified to immunize rabbit in order to produce a polyclonal antibody. Fluorescein isothiocyanate (FITC) was attached to the antibody and the prepared complex was applied to screening of Brucella spp., as an IF test. Polyclonal antibody obtained from the sera of immunized rabbit with the purified OMP16 showed a strong reactivity in ELISA and properly identified the B. abortus strain in IF test. So, this high titer of antibody could be helpful for tracing of B. abortus via laboratory diagnostic tests. [ABSTRACT FROM AUTHOR]

Serological data are one of the primary sources of information for disease monitoring in wildlife. However, the duration of the seropositive status of exposed individuals is almost always unknown for many free-ranging host species. Directly estimating rates of antibody loss typically requires difficult longitudinal sampling of individuals following seroconversion. Instead, we propose a Bayesian statistical approach linking age and serological data to a mechanistic epidemiological model to infer brucellosis infection, the probability of antibody loss, and recovery rates of elk ( Cervus canadensis) in the Greater Yellowstone Ecosystem. We found that seroprevalence declined above the age of ten, with no evidence of disease-induced mortality. The probability of antibody loss was estimated to be 0.70 per year after a five-year period of seropositivity and the basic reproduction number for brucellosis to 2.13. Our results suggest that individuals are unlikely to become re-infected because models with this mechanism were unable to reproduce a significant decline in seroprevalence in older individuals. This study highlights the possible implications of antibody loss, which could bias our estimation of critical epidemiological parameters for wildlife disease management based on serological data. [ABSTRACT FROM AUTHOR]

The innate immune system is essential for the detection and elimination of bacterial pathogens. Upon inflammasome activation, caspase-1 cleaves pro-IL-1β and pro-IL-18 to their mature forms IL-1β and IL-18, respectively, and the cell undergoes inflammatory death termed pyroptosis. Here, we reviewed recent findings demonstrating that Brucella abortus ligands activate NLRP3 and AIM2 inflammasomes which lead to control of infection. This protective effect is due to the inflammatory response caused by IL-1β and IL-18 rather than cell death. Brucella DNA is sensed by AIM2 and bacteria-induced mitochondrial reactive oxygen species is detected by NLRP3. However, deregulation of pro-inflammatory cytokine production can lead to immunopathology. Nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder termed neurobrucellosis. Herein, we discuss the mechanism of caspase-1 activation and IL-1β secretion in glial cells infected with B. abortus. Our results demonstrate that the ASC inflammasome is indispensable for inducing the activation of caspase-1 and secretion of IL-1β upon infection of astrocytes and microglia with Brucella. Moreover, our results demonstrate that secretion of IL-1β by Brucella-infected glial cells depends on NLRP3 and AIM2 and leads to neurobrucellosis. Further, the inhibition of the host cell inflammasome as an immune evasion strategy has been described for bacterial pathogens. We discuss here that the bacterial type IV secretion system VirB is required for inflammasome activation in host cells during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes mainly NLRP3 and AIM2 that collectively orchestrate a robust caspase-1 activation and pro-inflammatory response. [ABSTRACT FROM AUTHOR]

Brucellosis is a significant zoonotic disease that is emerging or reemerging in many parts of the world. This study was carried out to diagnose and investigate the pathological lesions associated with Brucella abortus in the fetuses and placenta of naturally infected dromedary camels. For diagnosis, the blood samples from infected dams were subjected to Rose Bengal Plate Test (RBPT) and blood PCR. The placental and fetal lung tissue samples were subjected to pathological examination and PCR. Nine cases of abortions and one case of early neonatal mortality was reported in the infected camels. The hematological parameters in infected dams revealed anemia and leukocytosis. The important pathological lesions in the infected fetuses were subcutaneous edema, moderate amount of serohemorrhagic fluid in the body cavities, interstitial pneumonia, degenerative changes in the liver, severe congestion with mononuclear infiltration in the kidney, and congestion in other visceral organs. The placentas were thickened, edematous, and showed necrosis along with mononuclear infiltration in histopathology. The RBPT and PCR for placental and fetal lung tissues detected all ten cases positive for B. abortus infection. However, blood PCR was positive only in two pregnant camels. The findings of the study indicated that B. abortus infection in pregnant dromedary camels causes necrotizing placentitis and fetal pneumonia resulting into abortion during mid to last trimester of pregnancy. The RBPT was found to play an important role in early serological diagnosis, whereas PCR was useful in confirmatory diagnosis of brucellosis from placental and fetal lung tissue samples. [ABSTRACT FROM AUTHOR]

We studied the effect of the organoselenium compound 2,6-dipyridinium-9-selenium-bicyclo[ 3,3,1]nonan dibromide (974zh) on the severity of pathological changes in the organs of experimental animals immunized with live tularemia and brucellosis vaccines. It was found that 974zh reduced reactogenicity of vaccines for experimental animals. Our findings indicate the prospects for further studies of the effects of 974zh on the functional state of experimental animals. [ABSTRACT FROM AUTHOR]

The intracellular pathogen Brucella abortus ( B. abortus) survives and replicates inside host cells within the Brucella-containing vacuole, in which membrane contains a small GTPase Rab1. Here, we reported that Rab1 mediates B. abortus intracellular growth. Furthermore, B. abortus DnaK was identified to interact with Rab1 using GST pull-down and mass spectrometry analysis. This interaction was confirmed by co-immunoprecipitation and immunofluorescence. Through DnaK-CyaA fusion protein translocation assay and immunofluorescence confocal microscopy, the B. abortus DnaK was proved to be a virB-dependent translocated substrate. [ABSTRACT FROM AUTHOR]

Brucellae are Gram-negative intracellular bacteria that cause an important zoonotic disease called brucellosis. The animal vaccines are available but have disadvantage of causing abortions in a proportion of pregnant animals. The animal vaccines are also pathogenic to humans. Recent trend in vaccine design has shifted to epitope-based vaccines that are safe and specific. In this study, efforts were made to identify MHC-I- and MHC-II-restricted T cell epitopes of Brucella abortus and evaluate their vaccine potential in mice. The peptides were designed using online available immunoinformatics tools, and five MHC-I- and one MHC-II-restricted T cell peptides were selected on the basis of their ability to produce interferon gamma (IFN-γ) in in vivo studies. The selected peptides were co-administered with poly dl-lactide-co-glycolide (PLG) microparticles and evaluated for immunogenicity and protection in BALB/c mice. Mice immunized with peptides either entrapped in PLG microparticles (EPLG-Pep) or adsorbed on PLG particles (APLG-Pep) showed significantly higher splenocyte proliferation and IFN-γ generation to all selected peptides than the mice immunized with corresponding irrelevant peptides formulated PLG microparticles or phosphate-buffered saline (PBS). A significant protection compared to PBS control was also observed in EPLG-Pep and APLG-Pep groups. A plasmid DNA vaccine construct (pVaxPep) for peptides encoding DNA sequences was generated and injected to mice by in vivo electroporation. Significant protection was observed (1.66 protection units) when compared with PBS and empty vector control group animals. Overall, the MHC-I and MHC-II peptides identified in this study are immunogenic and protective in mouse model and support the feasibility of peptide-based vaccine for brucellosis. [ABSTRACT FROM AUTHOR]

Brucella abortus lipopolysaccharide (LPS) has less toxicity and no pyrogenic properties in comparison with other bacterial LPS. It is a toll-like receptor 4 agonist and has been shown to have the potential use as a vaccine adjuvant. In this study, the immunostimulatory properties of LPS from smooth and rough strains of B. abortus (S19 and RB51) as adjuvants were investigated for the human papillomavirus type 16 (HPV16) L1 virus-like particles (L1VLPs) vaccines. C57BL/6 mice were immunized subcutaneously three times either with HPV-16 L1VLPs alone, or in combination with smooth LPS (S-LPS), rough LPS (R-LPS), aluminum hydroxide or a mixture of them as adjuvant. The humoral immunity was evaluated by measuring the specific and total IgG levels, and also the T-cell immune response of mice was evaluated by measuring different cytokines such as IFN-γ, TNF-α, IL-4, IL-10 and IL-17. Results showed that serum anti-HPV16 L1VLP IgG antibody titers was significantly higher in mice immunized with a combination of VLPs and R-LPS or S-LPS compared with other immunized groups. Co-administration of HPV-16 L1VLPs with R-LPS elicited the highest levels of splenocytes cytokines (IFN-γ, IL-4, IL-17 and TNF-α) and also effectively induced improvement of a Th1-type cytokine response characterized with a high ratio of IFN-γ/IL-10. The data indicate that B. abortus LPS particularly RB51-LPS enhances the immune responses to HPV-16 L1VLPs and suggests its potential as an adjuvant for the development of a potent prophylactic HPV vaccine and other candidate vaccines. [ABSTRACT FROM AUTHOR]

Brucellosis is an infectious and contagious disease that profoundly impacts public health. However, in many countries, disease prevention is restricted to the vaccination of calves, and there is no prophylactic strategy for pregnant heifers and cows. The aim of this study was to evaluate the safety of the rough strain vaccine against brucellosis in pregnant cattle. Crossbred cows ( N = 96) at three gestational periods (early, mid, or late pregnancy) were randomly allocated into the vaccine treatment group or to the control group. We then compared the percentage of pregnancies reaching full term, live calves 60 days after delivery, and seropositive calves. There was no effect of vaccination in any of the gestational periods on the evaluation endpoints. In conclusion, vaccination against brucellosis with the rough strain is safe for pregnant cattle at all gestational periods. [ABSTRACT FROM AUTHOR]

Bovine brucellosis is endemic in many parts of the world including India. The disease diagnosis and surveillance are usually carried out by serological tests, which however have drawbacks. This study was undertaken to evaluate the potential of real-time PCR (RT-PCR) targeting bcsp31 gene for surveillance of bovine brucellosis. A total of 461 samples, which included 408 stored serum and 53 prospective blood samples, were used. It was found that 33 (7.15 %) samples were positive by RT-PCR, whereas 149 (32.32 %) and 132 (28.63 %) were positive by Rose Bengal plate test (RBPT) or standard agglutination test (STAT), respectively. The results of this study suggest that RT-PCR targeting bcsp31 gene carried out on DNA extracted from serum or blood may not be a suitable method for surveillance of brucellosis in bovines. [ABSTRACT FROM AUTHOR]

Brucella abortus is a gram-negative, facultative intracellular pathogen that causes brucellosis, a chronic zoonotic disease resulting in abortion in pregnant cattle and undulant fever in humans. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, plays important metabolic roles in aerobic energy producing pathways and in malate shuttle. In this study, the MDH-encoding gene for malate dehydrogenase mdh of B. abortus S2308 was cloned, sequenced and expressed. Western blot analysis demonstrated that MDH is an immunogenic membrane-associated protein. In addition, recombinant MDH showed sero-reactivity with 30 individual bovine B. abortus-positive sera by enzyme-linked immunosorbent assay, indicates that MDH may be used as a candidate marker for sero-diagnosis of brucellosis. Furthermore, MDH exhibits fibronectin and plasminogen-binding ability in immunoblotting assay. Inhibition assays on HeLa cells demonstrated that rabbit anti-serum against MDH significantly reduced both bacterial adherence and invasion abilities ( p < 0.05), suggesting that MDH play a role in B. abortus colonization. Our results indicated that MDH is not only an immunogenic protein, but is also related to bacterial pathogenesis and may act as a new virulent factor, which will benefit for further understanding the MDH's roles in B. abortus metabolism, pathogenesis and immunity. [ABSTRACT FROM AUTHOR]

To evaluate the prevalence and possible risk factors for brucellosis caused by Brucella abortus in water buffaloes in the state of Pará, Brazil, 3,917 female buffalo serum samples from pregnant and non-pregnant animals were examined: 2,809 from Marajó Island and 1,108 from the mainland. The buffered acidified plate antigen (BAPA) screening test positively diagnosed 4.8 % (188/3,917) of the animals with brucellosis, and the 2-mercaptoethanol (2-ME) confirmatory test affirmed 95.7 % (180/188) of the results. The brucellosis prevalence was 4.17 times greater in mainland animals than on Marajó Island, with the highest prevalence in Tailândia (11.30 %) and Paragominas (12.38 %). Brucellosis seroprevalence was significantly influenced ( p < 0.05) by reproductive status, with pregnant females being most vulnerable. These results demonstrate that brucellosis infection is active in the Brazilian region containing the largest buffalo population and that this disease poses a threat to public health and buffalo production in Pará. [ABSTRACT FROM AUTHOR]

Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific ( Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk ( n = 5), aborted fetuses ( n = 13), and vaginal swabs ( n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan. [ABSTRACT FROM AUTHOR]

Water buffalo industry has become a profitable activity worldwide, including the Northeast of Argentina (NEA). However, research on diseases affecting this species is scarce. The aim of the present study was to detect antibodies against Brucella abortus, Leptospira spp., Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. in 500 water buffalo cows from five ranches (100 animals each) in the NEA. Serum samples were tested for B. abortus by fluorescence polarization assay, Leptospira spp. by microagglutination test, and N. caninum, T. gondii, and Sarcocystis spp. by indirect fluorescent antibody tests. Overall, the proportion of seropositive animals was 6.4, 22.2, 42.2, 25.4, and 50.8 % for brucellosis, leptospirosis, neosporosis, toxoplasmosis, and sarcocystosis, respectively. The proportion of seropositive animals for all diseases was statistically different among herds ( p < 0.05). Statistical differences were also detected among age groups for brucellosis and neosporosis ( p < 0.05). The detection of specific antibodies to B. abortus, Leptospira spp., and several Apicomplexa protozoans in water buffaloes in the NEA is reported in this study. [ABSTRACT FROM AUTHOR]

Brucellosis is a worldwide zoonotic disease. No Brucella vaccine is available for use in humans, and existing animal vaccines have limitations. There is a need to develop a safe and effective vaccine against human and animal brucellosis. In the present study, we generated recombinant cysteine synthase A (rCysK) of Brucella abortus in Escherichia coli and purified it up to homogeneity by metal affinity chromatography. The immunogenicity and protective efficacy of purified rCysK were evaluated in BALB/c mice with Freund's adjuvant, aluminium hydroxide gel or without any adjuvant. High titres of anti-rCysK IgG antibody predominated by IgG1 were observed in all immunized mice. After stimulation with rCysK, the spleen lymphocytes of mice immunized with CysK formulated with aluminium hydroxide gel produced significant levels of IFN-γ. Protection against challenge with virulent B. abortus strain 544 was determined in BALB/c mice, and significant protection was observed in all CysK immunized groups when compared with PBS control. Among all the CysK vaccine groups, comparatively better protection was observed in mice immunized with aluminium hydroxide gel (1.064 units of protection). Overall, the results of the study suggest that rCysK induces primarily Th2 type of immune response and provides partial protection against B. abortus challenge. [ABSTRACT FROM AUTHOR]

Brucellosis is an important zoonotic disease caused primarily by the bacterial pathogens Brucella melitensis and B. abortus. The pathogens cause debilitating febrile illness that can progress into a long-lasting disease with severe complications in humans. Understanding the mechanisms by which the host immune system responds to the infection will provide important information on the pathogenesis and development of differential diagnostic assays. In this study, a protein microarray was used to evaluate the antibody responses of brucellosis patients at different infection stages. A total of 107 outer membrane proteins, surface-exposed or secreted proteins, and known or putative virulence-associated proteins of B. melitensis were successfully expressed in Escherichia coli and used to fabricate the protein microarray. Then, 99 serum samples from acute, chronic, primary infection, or relapse brucellosis patients were probed with the protein microarray. Antibodies to 66 of the proteins were detected at least in one serum sample. Among the antigens, the combination of BMEII0318, BMEII0513, BMEI0748, and BMEII1116 could be used as serodiagnostic antigens for brucellosis. Patients at different infection stages show distinct antibody profiles. The numbers of antibodies in the relapse patients were superior to those in the primary infection patients, and the response magnitude of antibodies in the chronic infection patients was higher than those in the acute brucellosis patients. The sustained and differential antibody profiles of patients at different infection stages have implications for the development of new serological methods for the accurate diagnosis of human brucellosis, and contribute to a more detailed understanding of the pathogenesis of chronic brucellosis. [ABSTRACT FROM AUTHOR]