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Scientific Reports May 2020Brucella is alpha-2 Proteobacteria mainly responsible for multi-factorial bacterial zoonotic disease brucellosis with low concentration (10-100 CFU) required to...
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 10 to 10 cells mL and limit of detection at 10 cells mL by employing polyclonal rabbit IgG (capture antibody, 10 µg mL) and mice IgG (detection antibody, 50 µg mL) antibody for its detection. Surface Plasmon Resonance evaluated the interaction of detection antibody with whole cell spiked serum samples at LOD of 10 cells mL 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 (K) and Maximum Binding Capacity (B) 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.
Topics: Animals; Antibodies, Bacterial; Antigens, Bacterial; Brucella abortus; Brucella melitensis; Brucellosis; Female; Immunoassay; Immunologic Tests; Mice; Mice, Inbred BALB C; Rabbits
PubMed: 32444793
DOI: 10.1038/s41598-020-65347-9 -
Microbial Genomics Nov 2020Ovine and caprine brucellosis, caused by , is one of the world's most widespread zoonoses and is a major cause of economic losses in domestic ruminant production. In...
Ovine and caprine brucellosis, caused by , is one of the world's most widespread zoonoses and is a major cause of economic losses in domestic ruminant production. In Italy, the disease remains endemic in several southern provinces, despite an ongoing brucellosis eradication programme. In this study, we used whole-genome sequencing to detail the genetic diversity of circulating strains, and to examine the origins of the predominant sub-lineages of in Italy. We reconstructed a global phylogeny of , strengthened by 339 new whole-genome sequences, from Italian isolates collected from 2011 to 2018 as part of a national livestock surveillance programme. All Italian strains belonged to the West Mediterranean lineage, which further divided into two major clades that diverged roughly between the 5th and 7th centuries. We observed that Sicily serves as a brucellosis burden hotspot, giving rise to several distinct sub-lineages. More than 20 putative outbreak clusters of ovine and caprine brucellosis were identified, several of which persisted over the 8 year survey period despite an aggressive brucellosis eradication campaign. While the outbreaks in Central and Northern Italy were generally associated with introductions of single clones of and their subsequent dissemination within neighbouring territories, we observed weak geographical segregation of genotypes in the southern regions. Biovar determination, recommended in routine analysis of all strains by the World Organisation for Animal Health (OIE), could not discriminate among the four main global clades. This demonstrates a need for updating the guidelines used for monitoring transmission and spread, both at the national and international level, and to include whole-genome-based typing as the principal method for identification and tracing of brucellosis outbreaks.
Topics: Animals; Brucella melitensis; Brucellosis; Cattle; Cattle Diseases; Genetic Variation; Genome, Bacterial; Goat Diseases; Goats; Humans; Italy; Minisatellite Repeats; Multilocus Sequence Typing; Phylogeny; Sheep; Whole Genome Sequencing
PubMed: 33030422
DOI: 10.1099/mgen.0.000446 -
BMC Microbiology Sep 2014Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate...
BACKGROUND
Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate inside the cells. Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a worldwide extended chronic zoonosis. Following invasion, Brucella abortus is found in a vacuole that interacts first with various endosomal compartments and then with endoplasmic reticulum sub-compartments. Brucella establishes its replication niche in ER-derived vesicles. In the past, it has been proposed that B. abortus passed through the macroautophagy pathway before reaching its niche of replication. However, recent experiments provided evidence that the classical macroautophagy pathway was not involved in the intracellular trafficking and the replication of B. abortus in bone marrow-derived macrophages and in HeLa cells. In contrast, another study showed that macroautophagy favoured the survival and the replication of Brucella melitensis in infected RAW264.7 macrophages. This raises the possibility that B. abortus and B. melitensis followed different intracellular pathways before replicating. In the present work, we have addressed this issue by comparing the replication rate of B. abortus and B. melitensis in embryonic fibroblasts derived from wild-type and Atg5-/- mice, Atg5 being a core component of the canonical macroautophagic pathway.
RESULTS
Our results indicate that both B. abortus S2308 and B. melitensis 16M strains are able to invade and replicate in Atg5-deficient fibroblasts, suggesting that the canonical Atg5-dependent macroautophagic pathway is dispensable for Brucella replication. The number of viable bacteria was even slightly higher in Atg5-/- fibroblasts than in wild-type fibroblasts. This increase could be due to a more efficient uptake or to a better survival rate of bacteria before the beginning of the replication in Atg5-deficient cells as compared to wild-type cells. Moreover, our data show that the infection with B. abortus or with B. melitensis does not stimulate neither the conversion of LC3-I to LC3-II nor the membrane recruitment of LC3 onto the BCV.
CONCLUSION
Our study suggests that like Brucella abortus, Brucella melitensis does not subvert the canonical macroautophagy to reach its replicative niche or to stimulate its replication.
Topics: Animals; Autophagy; Autophagy-Related Protein 5; Brucella abortus; Brucella melitensis; Fibroblasts; Mice, Knockout; Microtubule-Associated Proteins
PubMed: 25179110
DOI: 10.1186/s12866-014-0223-5 -
Journal of Veterinary Science Sep 2019is an intracellular pathogen that invades a host and settles in its immune cells; however, the mechanism of its intracellular survival is unclear. Modification of small...
is an intracellular pathogen that invades a host and settles in its immune cells; however, the mechanism of its intracellular survival is unclear. Modification of small ubiquitin-related modifier (SUMO) occurs in many cellular activities. E2 conjugating enzyme 9 (Ubc9) is the only reported ubiquitin-conjugating enzyme that links the SUMO molecule with a target protein. 's intracellular survival mechanism has not been studied with respect to SUMO-related proteins and Ubc9. Therefore, to investigate the relationship between 16M and SUMO, we constructed plasmids and cells lines suitable for overexpression and knockdown of SUMO1 and Ubc9 genes. 16M activated SUMO1/Ubc9 expression in a time-dependent manner, and 16M intracellular survival was inhibited by SUMO1/Ubc9 overexpression and promoted by SUMO1/Ubc9 depletion. In macrophages, 16M-dependent apoptosis and immune factors were induced by SUMO1/Ubc9 overexpression and restricted by SUMO1/Ubc9 depletion. We noted no effect on the expressions of SUMO1 and Ubc9 in 16M lipopolysaccharide-prestimulated mouse RAW264.7 macrophages. Additionally, intracellular survival of the 16M△VirB2 mutant was lower than that of 16M ( < 0.05). VirB2 can affect expression levels of Ubc9, thereby increasing intracellular survival of in macrophages at the late stage of infection. Collectively, our results demonstrate that 16M may use the VirB IV secretion system of to interact with SUMO-related proteins during infection of host cells, which interferes with SUMO function and promotes pathogen survival in host cells.
Topics: Animals; Brucella melitensis; Mice; RAW 264.7 Cells; SUMO-1 Protein; Ubiquitin-Conjugating Enzymes
PubMed: 31565897
DOI: 10.4142/jvs.2019.20.e54 -
Infectious Diseases of Poverty Mar 2021The prevalence of human brucellosis in Qinghai Province of China has been increasing rapidly, with confirmed cases distributed across 31 counties. However, the...
BACKGROUND
The prevalence of human brucellosis in Qinghai Province of China has been increasing rapidly, with confirmed cases distributed across 31 counties. However, the epidemiology of brucellosis transmission has not been fully elucidated. To characterize the infecting strains isolated from humans, multiple-locus variable-number tandem repeats analysis (MLVA) and whole-genome single-nucleotide polymorphism (SNP)-based approaches were employed.
METHODS
Strains were isolated from two males blood cultures that were confirmed Brucella melitensis positive following biotyping and MLVA. Genomic DNA was extracted from these two strains, and whole-genome sequencing was performed. Next, SNP-based phylogenetic analysis was performed to compare the two strains to 94 B. melitensis strains (complete genome and draft genome) retrieved from online databases.
RESULTS
The two Brucella isolates were identified as B. melitensis biovar 3 (QH2019001 and QH2019005) following conventional biotyping and were found to have differences in their variable number tandem repeats (VNTRs) using MLVA-16. Phylogenetic examination assigned the 96 strains to five genotype groups, with QH2019001 and QH2019005 assigned to the same group, but different subgroups. Moreover, the QH2019005 strain was assigned to a new subgenotype, IIj, within genotype II. These findings were then combined to determine the geographic origin of the two Brucella strains.
CONCLUSIONS
Utilizing a whole-genome SNP-based approach enabled differences between the two B. melitensis strains to be more clearly resolved, and facilitated the elucidation of their different evolutionary histories. This approach also revealed that QH2019005 is a member of a new subgenotype (IIj) with an ancient origin in the eastern Mediterranean Sea.
Topics: Brucella melitensis; Brucellosis; China; Genotype; Humans; Male; Minisatellite Repeats; Multilocus Sequence Typing; Phylogeny
PubMed: 33771234
DOI: 10.1186/s40249-021-00829-0 -
Frontiers in Immunology 2022Brucellosis, caused by spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An...
Brucellosis, caused by spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, biovar 3 and compare it with those expressed by M5-90. The higher level of virulence for biovar 3 compared to M5-90 was validated and . A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to M5-90 and biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in biovar 3 was 8-fold and 2-fold higher than in M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.
Topics: ATP-Binding Cassette Transporters; Animals; Bacterial Proteins; Brucella melitensis; Brucellosis; Proteome; Proteomics; Thiamine; Virulence Factors
PubMed: 35928811
DOI: 10.3389/fimmu.2022.929040 -
Asian Pacific Journal of Tropical... Jan 2012To evaluate simultaneous detection and differentiates of Brucella abortus (B. abortus) and Brucella melitensis (B. melitensis) through the combinatorial PCR method.
OBJECTIVE
To evaluate simultaneous detection and differentiates of Brucella abortus (B. abortus) and Brucella melitensis (B. melitensis) through the combinatorial PCR method.
METHODS
This study was designed using three primers that could simultaneously identify and differentiate two major species of pathogenic Brucella in humans and animals. Identification and differentiation of each species using the size of the PCR product were determined. To determine the specificity of the method, bacteria close to the genus Brucella were used. Finally, to confirm PCR products, In addition to the products sequence, RFLP was performed on PCR products using restriction enzymes.
RESULTS
The method of optimized combinatorial PCR in this study could simultaneously detect and differentiate B. abortus and B. melitensis with high specificity and sensitivity in clinical samples. Differentiation of species is based on the resulting bands; therefore, the band 494 bp for B. abortus and 733 bp for B. melitensis were obtained. RFLP and sequencing results confirmed PCR results.
CONCLUSIONS
The results of this study shows that without routine diagnostic methods such as culture and serology tests, using the molecular method of combinatorial PCR, important species of Brucella can be simultaneously identified and differentiated in clinical samples.
Topics: Animals; Base Sequence; Brucella abortus; Brucella melitensis; Brucellosis; DNA Primers; DNA, Bacterial; Humans; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Sensitivity and Specificity; Species Specificity
PubMed: 22182638
DOI: 10.1016/S1995-7645(11)60239-3 -
Genetic characterization of Brucella melitensis and Brucella abortus geographical clusters in Italy.Veterinaria Italiana 2015The genetic diversity of Brucella melitensis and Brucella abortus strains isolated in 199 cattle and sheep from 156 brucellosis outbreaks which occurred in 8 regions of...
The genetic diversity of Brucella melitensis and Brucella abortus strains isolated in 199 cattle and sheep from 156 brucellosis outbreaks which occurred in 8 regions of Southern Italy in 2011, was determined using a Multiple-Locus Variable Number of Tandem Repeats Analysis approach. The existence of possible genetic clusters was verified through a hierarchical cluster analysis based on 'single link', which is closely related to the minimum spanning tree. The Hamming weighted distance matrix was adopted in the analysis. All calculations were performed using R and the additional libraries phangorn and Cluster. For a number of clusters, ranging from 2 to 15, the average silhouette width was calculated. The number of clusters adopted was identified according to the maximum average silhouette width. For B. abortus and B. melitensis, 6 and 11 genetic clusters were identified, respectively. Three out of 6 B. abortus clusters included the 96.7% of all B. abortus isolates. Clusters were clearly geographically separated, and this highlighted the known epidemiological links among them. Brucella melitensis genotypes resulted more heterogeneous; the 3 more representative genetic clusters included 79.7% of all B. melitensis isolates. A clear geographical clusterization of genotypes is recognizable only for 1 cluster, whereas the others are more widespread across Southern Italy. The genetic characterization of Brucella strains isolated from animals may be a useful tool to better understand the epidemiology and dissemination patterns of this pathogen through host populations.
Topics: Animals; Brucella abortus; Brucella melitensis; Brucellosis, Bovine; Cattle; Cluster Analysis; Genotype; Italy; Sheep; Sheep Diseases
PubMed: 26455376
DOI: 10.12834/VetIt.312.1218.3 -
Infection and Immunity Oct 2020, the causative agent of brucellosis, is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals, including humans. The twin-arginine...
, the causative agent of brucellosis, is a stealthy intracellular pathogen that is highly pathogenic to a range of mammals, including humans. The twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane and has been implicated in virulence in many bacterial pathogens. However, the roles of the Tat system and related substrates in remain unclear. We report here that disruption of Tat increases the sensitivity of M28 to the membrane stressor sodium dodecyl sulfate (SDS), indicating cell envelope defects, as well as to EDTA. In addition, mutating Tat renders M28 bacteria more sensitive to oxidative stress caused by HO Further, loss of Tat significantly attenuates infection in murine macrophages Using a mouse model for persistent infection, we demonstrate that Tat is required for full virulence of M28. Genome-wide prediction combined with an amidase reporter assay indicates that at least 23 proteins are authentic Tat substrates, and they are functionally categorized into solute-binding proteins, oxidoreductases, cell envelope biosynthesis enzymes, and others. A comprehensive deletion study revealed that 6 substrates contribute significantly to virulence, including an l,d-transpeptidase, an ABC transporter solute-binding protein, and a methionine sulfoxide reductase. Collectively, our work establishes that the Tat pathway plays a critical role in virulence.
Topics: Animals; Bacterial Proteins; Brucella melitensis; Brucellosis; Mice; Stress, Physiological; Twin-Arginine-Translocation System; Virulence
PubMed: 32778612
DOI: 10.1128/IAI.00389-20 -
PLoS Neglected Tropical Diseases Mar 2022Brucellosis is an infectious disease caused by bacteria of the genus Brucella. Although it is the most common zoonosis worldwide, there are increasing reports of drug...
BACKGROUND
Brucellosis is an infectious disease caused by bacteria of the genus Brucella. Although it is the most common zoonosis worldwide, there are increasing reports of drug resistance and cases of relapse after long term treatment with the existing drugs of choice. This study therefore aims at identifying possible natural inhibitors of Brucella melitensis Methionyl-tRNA synthetase through an in-silico approach.
METHODS
Using PyRx 0.8 virtual screening software, the target was docked against a library of natural compounds obtained from edible African plants. The compound, 2-({3-[(3,5-dichlorobenzyl) amino] propyl} amino) quinolin-4(1H)-one (OOU) which is a co-crystallized ligand with the target was used as the reference compound. Screening of the molecular descriptors of the compounds for bioavailability, pharmacokinetic properties, and bioactivity was performed using the SWISSADME, pkCSM, and Molinspiration web servers respectively. The Fpocket and PLIP webservers were used to perform the analyses of the binding pockets and the protein ligand interactions. Analysis of the time-resolved trajectories of the Apo and Holo forms of the target was performed using the Galaxy and MDWeb servers.
RESULTS
The lead compounds, Strophanthidin and Isopteropodin are present in Corchorus olitorius and Uncaria tomentosa (Cat's-claw) plants respectively. Isopteropodin had a binding affinity score of -8.9 kcal / ml with the target and had 17 anti-correlating residues in Pocket 1 after molecular dynamics simulation. The complex formed by Isopteropodin and the target had a total RMSD of 4.408 and a total RMSF of 9.8067. However, Strophanthidin formed 3 hydrogen bonds with the target at ILE21, GLY262 and LEU294, and induced a total RMSF of 5.4541 at Pocket 1.
CONCLUSION
Overall, Isopteropodin and Strophanthidin were found to be better drug candidates than OOU and they showed potentials to inhibit the Brucella melitensis Methionyl-tRNA synthetase at Pocket 1, hence abilities to treat brucellosis. In-vivo and in-vitro investigations are needed to further evaluate the efficacy and toxicity of the lead compounds.
Topics: Anti-Bacterial Agents; Brucella melitensis; Ligands; Methionine-tRNA Ligase; Molecular Dynamics Simulation
PubMed: 35312681
DOI: 10.1371/journal.pntd.0009799