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Archives of Razi Institute Jun 2019Brucellosis is a zoonotic infection that is associated with fever in humans and abortion in animals. The agent of this disease is a facultative intracellular...
Brucellosis is a zoonotic infection that is associated with fever in humans and abortion in animals. The agent of this disease is a facultative intracellular gram-negative coccobacillus called Brucella. There are six classic species, including B. abortus, B. melitensis, B. suis, B. canis, B. neotomae, and B. ovis. In recent years, four new species have been reported, including Brucella ceti, B. microti, B. pinnipedialis, and B. inopinata. Human disease causes hygienic and economic losses, including inactivity of workforces in the community and high cost of treatment. The disease also causes catastrophic losses in the livestock industry. There is no effective vaccine against human brucellosis. Hence, attempts to prevent human infection with Brucella are focused on preventative measures, including control of infection in livestock, which lead to a reduction in its incidence in humans. The common methods for diagnosis of this disease are serologic methods including Rose Bengal, Wright -2 ME and the ring test. B. abortus strain S99 is used to produce these diagnostic antigens. The production of these antigens requires the presence of a well-characterized seed with full identity. The aim of this work was confirmation of the identity of B. abortus S99 by phage typing, AMOS and multiplex PCR techniques. Therefore, it is essential to carry out the identification of the strains used as seed for the production of the brucellosis diagnostic antigens. In this project, B. abortus strain 99 was supplied by the bacterial collection of the Brucellosis Department of Razi Vaccine and Serum Research Institute. Then, the main aim of the present study was the confirmation of the seed identity by doing the tests through the standard phage typing method, AMOS PCR and multiplex PCR (Brucladder) methods. Results were in support of the identity of the studied strain, and the molecular methods could also be used as the sensitive approaches for validation of antigenic seed.
Topics: Animals; Bacteriophage Typing; Brucella abortus; Brucellosis; Brucellosis, Bovine; Cattle; Humans; Polymerase Chain Reaction
PubMed: 31232562
DOI: 10.22092/ari.2019.123507.1255 -
Archives of Razi Institute Oct 2022Brucellosis is an important contagious disease affecting most domestic and mature animals. Since the impact of IL-1β in invasion and survival remains elusive, the...
Brucellosis is an important contagious disease affecting most domestic and mature animals. Since the impact of IL-1β in invasion and survival remains elusive, the current study sought to elucidate the actual roles of these potent cytokines in the modulation of the initial immune response to infection. Therefore, this study aimed to detect in the placenta of aborted women and cows and estimate the expression of the interleukin 1β () gene associated with immune response mechanisms to infection. The detection of was performed by Rose Bengal Test (RBT) and Polymerase Chain Reaction based gene (AlkB-PCR) in the sera and placenta samples of aborted women and cows, respectively. The overall percentage of infection was 13.1% and 5% as determined by RBT and -PCR in aborted women's sera and placentas, respectively. On the other hand, the overall percentage rates of infection in the sera and placentas from aborted cows were 30% and 11% as estimated by RBT and -PCR, respectively. The results of RBT demonstrated that the association between and abortion in cows was statistically significant. On the other hand, it was found that the association between and abortion in women was not significant. Moreover, according to the results of -based PCR, the association between and abortion was statistically significant in aborted cows, while it was not significant in aborted women. The sensitivity, specificity, and accuracy of RBT were calculated as 60.00, 53.85, and 54.55%, respectively. Moreover, positive and negative predictive values were reported as 14.33% and 91.28%, respectively. Regarding RBT for aborted cows, the sensitivity, specificity, and accuracy of the test were 81.82%, 57.78%, and 62.49%, respectively. The positive predictive value was reported as 32.08%, while the negative predictive value was reported as 92.88%. Quantitative PCR (qPCR) was carried out for the evaluation of Interleukin 1 Beta () gene expression. The qPCR result was presented as a fold change in gene expression. A significant increment of gene expression was observed in aborted women (114.905±99.661) and cows (22.454 ±18.528), compared to non-aborted women (4.953±5.564) and cows (2.033±1.845). Statistical comparison of gene expression between aborted women and cows illustrated a non-significant increment in gene expression in aborted women (114.905±99.661), compared to aborted cows (22.454 ±18.528).
Topics: Animals; Cattle; Female; Humans; Pregnancy; Abortion, Veterinary; Brucella abortus; Brucellosis; Brucellosis, Bovine; Cattle Diseases; Interleukin-1beta; Placenta; Rose Bengal; Abortion, Spontaneous
PubMed: 37123145
DOI: 10.22092/ARI.2022.358327.2192 -
Clinical and Vaccine Immunology : CVI Nov 2014Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain...
Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response.
Topics: ATP-Binding Cassette Transporters; Animals; Bacterial Proteins; Brucella Vaccine; Brucella abortus; Carrier Proteins; Cell Line; DNA Transposable Elements; Epithelial Cells; Female; Gene Deletion; Genetic Complementation Test; Humans; Injections, Intraperitoneal; Macrophages; Mice, Inbred BALB C; Mutagenesis, Insertional; Vaccination; Vaccines, Attenuated; Virulence; Virulence Factors
PubMed: 25253663
DOI: 10.1128/CVI.00164-14 -
Infection and Immunity Mar 2022Research on Brucella pathogenesis has focused primarily on its ability to cause persistent intracellular infection of the mononuclear phagocyte system. At these sites,...
Research on Brucella pathogenesis has focused primarily on its ability to cause persistent intracellular infection of the mononuclear phagocyte system. At these sites, Brucella abortus evades innate immunity, which results in low-level inflammation and chronic infection of phagocytes. In contrast, the host response in the placenta during infection is characterized by severe inflammation and extensive extracellular replication of B. abortus. Despite the importance of reproductive disease caused by Brucella infection, our knowledge of the mechanisms involved in placental inflammation and abortion is limited. To understand the immune responses specifically driving placental pathology, we modeled placental B. abortus infection in pregnant mice. B. abortus infection caused an increase in the production of tumor necrosis factor alpha (TNF-α), specifically in the placenta. We found that placental expression levels of and circulating TNF-α were dependent on the induction of endoplasmic reticulum stress and the B. abortus type IV secretion system (T4SS) effector protein VceC. Blockade of TNF-α reduced placental inflammation and improved fetal viability in mice. This work sheds light on a tissue-specific response of the placenta to B. abortus infection that may be important for bacterial transmission via abortion in the natural host species.
Topics: Animals; Brucella abortus; Brucellosis; Brucellosis, Bovine; Cattle; Female; Inflammation; Mice; Placenta; Pregnancy; Tumor Necrosis Factor-alpha
PubMed: 35100011
DOI: 10.1128/iai.00013-22 -
PloS One 2021Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several...
Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.
Topics: Animals; Brucella Vaccine; Brucella abortus; Brucellosis, Bovine; Cattle; Enzyme-Linked Immunosorbent Assay; Female; Fluorescence; Green Fluorescent Proteins; Mice; Multiplex Polymerase Chain Reaction; Vaccination
PubMed: 34807952
DOI: 10.1371/journal.pone.0260288 -
Journal of Bacteriology Jun 2018The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in is linked to a variety...
The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in is linked to a variety of important activities, including proper cellular morphology, mRNA transcript levels, and virulence. Deletion of in led to a small-colony phenotype when the bacteria were grown on agar medium, as well as to significant aberrations in the morphology of the bacterial cell as evidenced by electron microscopy. Additionally, compared to the parental strain, the Δ strain was significantly attenuated in both macrophage and mouse models of infection. The Δ strain also showed increased sensitivities to several -applied stressors, including bile acid, hydrogen peroxide, SDS, and paraquat. Transcriptomic analysis revealed that a multitude of mRNA transcripts are dysregulated in the Δ strain, and many of the identified mRNAs encode proteins involved in metabolism, nutrient transport, transcriptional regulation, and flagellum synthesis. We subsequently constructed gene deletion strains of the most highly dysregulated systems, and several of the YbeY-linked gene deletion strains exhibited defects in the ability of the bacteria to survive and replicate in primary murine macrophages. Taken together, these data establish a clear role for YbeY in the biology and virulence of ; moreover, this work further illuminates the highly varied roles of this widely conserved endoribonuclease in bacteria. spp. are highly efficient bacterial pathogens of animals and humans, causing significant morbidity and economic loss worldwide, and relapse of disease often occurs following antibiotic treatment of human brucellosis. As such, novel therapeutic strategies to combat infections are needed. Ribonucleases in the brucellae are understudied, and these enzymes represent elements that may be potential targets for future treatment approaches. The present work demonstrates the importance of the YbeY endoribonuclease for cellular morphology, efficient control of mRNA levels, and virulence in Overall, the results of this study advance our understanding of the critical roles of YbeY in the pathogenesis of the intracellular brucellae and expand our understanding of this highly conserved RNase.
Topics: Animals; Bacterial Proteins; Brucella abortus; Brucellosis; Endoribonucleases; Female; Gene Expression Regulation, Bacterial; Humans; Macrophages; Male; Mice; Mice, Inbred BALB C; Virulence
PubMed: 29632093
DOI: 10.1128/JB.00105-18 -
Viruses Jun 2017For decades, bacteriophages (phages) have been used for species identification in the diagnosis and epidemiology of brucellosis. Traditional phage typing is a...
For decades, bacteriophages (phages) have been used for species identification in the diagnosis and epidemiology of brucellosis. Traditional phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with pure culture and with mixed cultures of . and α-proteobacterial near neighbors that can be misidentified as spp., and . The addition of a simple short sample preparation step enabled the indirect phage-based detection of . in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live . in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types.
Topics: Alphaproteobacteria; Bacteriophages; Brucella abortus; Brucellosis; Humans; Nucleic Acid Amplification Techniques; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Specimen Handling; Virology
PubMed: 28604602
DOI: 10.3390/v9060144 -
Frontiers in Immunology 2022Endoplasmic reticulum (ER) stress plays a major role in several inflammatory disorders. ER stress induces the unfolded protein response (UPR), a conserved response...
Endoplasmic reticulum (ER) stress plays a major role in several inflammatory disorders. ER stress induces the unfolded protein response (UPR), a conserved response broadly associated with innate immunity and cell metabolic function in various scenarios. , an intracellular pathogen, triggers the UPR Stimulator of interferon genes (STING), an important regulator of macrophage metabolism during infection. However, whether ER stress pathways underlie macrophage metabolic function during infection remains to be elucidated. Here, we showed that the UPR sensor inositol-requiring enzyme 1α (IRE1α) is as an important component regulating macrophage immunometabolic function. In infection, IRE1α supports the macrophage inflammatory profile, favoring M1-like macrophages. IRE1α drives the macrophage metabolic reprogramming in infected macrophages, contributing to the reduced oxidative phosphorylation and increased glycolysis. This metabolic reprogramming is probably associated with the IRE1α-dependent expression and stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), an important molecule involved in cell metabolism that sustains the inflammatory profile in -infected macrophages. Accordingly, we demonstrated that IRE1α favors the generation of mitochondrial reactive oxygen species (mROS) which has been described as an HIF-1α stabilizing factor. Furthermore, in infected macrophages, IRE1α drives the production of nitric oxide and the release of IL-1β. Collectively, these data unravel a key mechanism linking the UPR and the immunometabolic regulation of macrophages in infection and highlight IRE1α as a central pathway regulating macrophage metabolic function during infectious diseases.
Topics: Animals; Cattle; Brucella abortus; Brucellosis, Bovine; Endoplasmic Reticulum Stress; Endoribonucleases; Macrophages; Protein Serine-Threonine Kinases
PubMed: 36660548
DOI: 10.3389/fimmu.2022.1063221 -
Frontiers in Immunology 2023Despite the importance of the respiratory route for transmission, the lung immune response to this pathogen is scarcely characterized. We investigated the role of the...
Despite the importance of the respiratory route for transmission, the lung immune response to this pathogen is scarcely characterized. We investigated the role of the cGAS/STING pathway of microbial DNA recognition in the control of respiratory infection. After infection, CFU numbers were significantly higher in alveolar macrophages (AM) and lung explants from STING KO mice than in samples from wild type (WT) mice, but no difference was observed for cGAS KO samples. CFU were also increased in WT AM and lung epithelial cells preincubated with the STING inhibitor H151. Several proinflammatory cytokines (TNF-α, IL-1β, IL-6, IP-10/CXCL10) were diminished in -infected lung explants and/or AM from STING KO mice and cGAS KO mice. These cytokines were also reduced in infected AM and lung epithelial cells pretreated with H151. After intratracheal infection with , STING KO mice exhibited increased CFU in lungs, spleen and liver, a reduced expression of IFN-β mRNA in lungs and spleen, and reduced levels of proinflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF) and lung homogenates. Increased lung CFU and reduced BALF cytokines were also observed in cGAS KO mice. In summary, the cGAS/STING pathway induces the production of proinflammatory cytokines after respiratory infection, which may contribute to the STING-dependent control of airborne brucellosis.
Topics: Animals; Mice; Cattle; Brucella abortus; Brucellosis; Cytokines; Brucellosis, Bovine; Nucleotidyltransferases
PubMed: 37261352
DOI: 10.3389/fimmu.2023.1116811 -
Frontiers in Immunology 2018induces an inflammatory response that stimulates the endocrine system resulting in the secretion of cortisol and dehydroepiandrosterone (DHEA). Osteoarticular...
induces an inflammatory response that stimulates the endocrine system resulting in the secretion of cortisol and dehydroepiandrosterone (DHEA). Osteoarticular brucellosis is the most common presentation of the active disease in humans, and we have previously demonstrated that infection inhibits osteoblast function. We aimed to evaluate the role of cortisol and DHEA on osteoblast during infection. infection induces apoptosis and inhibits osteoblast function. DHEA treatment reversed the effect of infection on osteoblast by increasing their proliferation, inhibiting osteoblast apoptosis, and reversing the inhibitory effect of on osteoblast differentiation and function. By contrast, cortisol increased the effect of infection. Cortisol regulates target genes by binding to the glucocorticoid receptor (GR). infection inhibited GRα expression. Cell responses to cortisol not only depend on GR expression but also on its intracellular bioavailability, that is, dependent on the activity of the isoenzymes 11β-hydroxysteroid dehydrogenase (HSD) type-1, 11β-HSD2 (which convert cortisone to cortisol and , respectively). Alterations in the expression of these isoenzymes in bone cells are associated with bone loss. infection increased 11β-HSD1 expression but had no effect on 11β-HSD2. DHEA reversed the inhibitory effect induced by infection on osteoblast matrix deposition in an estrogen receptor- and ERK1/2-dependent manner. We conclude that DHEA intervention improves osteoblast function during infection making it a potential candidate to ameliorate the osteoarticular symptoms of brucellosis.
Topics: Animals; Apoptosis; Biomarkers; Brucella abortus; Brucellosis, Bovine; Cattle; Cell Differentiation; Cell Line; Cell Proliferation; Dehydroepiandrosterone; Gene Expression; Mice; Microbial Viability; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Osteoblasts; Osteogenesis; Receptors, Estrogen; Receptors, Glucocorticoid
PubMed: 29434601
DOI: 10.3389/fimmu.2018.00088