National epidemiology of culture-confirmed brucellosis in Israel, 2004-2022.

Brucellosis, a global zoonosis, is endemic in Israel. We used a national database of culture-confirmed cases (2004-2022) to analyse the trends of brucellosis. Of 2,489 unique cases, 99.8% were bacteraemic, 64% involved males, and the mean age was 30.5 years. Brucella melitensis was the dominant species (99.6%). Most cases occurred among the Arab sector (84.9%) followed by the Jewish (8.5%) and Druze (5.5%) sectors. The average annual incidence rates overall and for the Arab, Druze, and Jewish sectors were 1.6/100,000, 6.6/100,000, 5.5/100,000, and 0.18/100,000, respectively. The annual incidence rates among the Arab (incidence rate ratio (IRR) = 36.4) and the Druze (IRR = 29.6) sectors were significantly higher than among the Jewish sector (p < 0.001). The highest incidence rates among the Arab sector occurred in the South District, peaking at 41.0/100,000 in 2012. The frequencies of B. melitensis isolated biotypes (biotype 1 - 69.1%, biotype 2 - 26.0%, and biotype 3 - 4.3%) differed from most Middle Eastern and European countries. A significant switch between the dominant biotypes was noted in the second half of the study period. Efforts for control and prevention should be sustained and guided by a One Health approach mindful of the differential trends and changing epidemiology.

Molecular epidemiological characteristics of osteoarthritis-associated Brucella melitensis in China: evidence from whole-genome sequencing-based analysis.

BACKGROUND: Brucellosis, developing complications including arthritis, spondylitis, sacroiliitis, and osteomyelitis, is one of the most common zoonotic diseases in the current world which causes economic losses to the livestock industry and is a great public health concern. Brucella melitensis are the main pathogen of brucellosis epidemics in China, most of which are located in northern China. However, there is limited knowledge about the epidemiology of osteoarthritis-associated brucellosis. This study was aimed to reveal the prevalence of osteoarthritis-associated brucellosis in Inner Mongolia and also to investigate the molecular characteristics of B. melitensis isolates. METHODS AND RESULTS: In 2018, the osteoarthritis symptoms of brucellosis in the Brucellosis department of a hospital in Inner Mongolia were investigated. Twenty osteoarthritis-associated B. melitensis strains, isolated from the inpatients in Inner Mongolia during 2013-2017, were subjected to whole genome sequencing. The multilocus sequence type (MLST) and core genome SNP (cgSNP) analysis were conducted to detect molecular epidemiological characteristics. The incidence of brucellosis osteoarthritis symptoms in males (85/120, 70.8%) was significantly higher than that in females (35/120, 29.2%), and the age of patients was concentrated between 41 and 60 years old. In silico analyses indicated ST8 was the prevalent sequence type and the transmission of osteoarthritis-associated B. melitensis among different geographical areas. All strains carry virulence genes, including cgs, lpsA, manCoAg, pgm, pmm, virB4, wbdA and wboA. CONCLUSION: Our study showed the close epidemiologically connection of osteoarthritis-associated B. melitensis strains in northern China. And ST8 was the prevalent sequence type which need our attention.

Prevalence of Brucella melitensis and Brucella abortus tetracyclines resistance: A systematic review and meta-analysis.

INTRODUCTION: Brucellosis is a zoonotic disease that can be transmitted from animals to humans. Brucellosis is caused by bacteria of the genus Brucella, which are typically transmitted through contact with infected animals, unpasteurized dairy products, or airborne pathogens. Tetracyclines (tetracycline and doxycycline) are antibiotics commonly used to treat brucellosis; however, antibiotic resistance has become a major concern. This study assessed the worldwide prevalence of tetracycline-resistant Brucella isolates. METHODS: A systematic search was conducted in Scopus, PubMed, Web of Science, and EMBASE using relevant keywords and Medical Subject Headings (MeSH) terms until August 13, 2022, to identify relevant studies for meta-analysis. A random effects model was used to estimate the proportion of resistance. Meta-regression analysis, subgroup analysis, and examination of outliers and influential studies were also performed. RESULTS: The prevalence rates of resistance to tetracycline and doxycycline were estimated to be 0.017 (95% confidence interval [CI], 0.009-0.035) and 0.017 (95%CI, 0.011-0.026), respectively, based on 51 studies conducted from 1983 to 2020. Both drugs showed increasing resistance over time (tetracycline: r = 0.077, P = 0.012; doxycycline: r = 0.059, P = 0.026). CONCLUSION: The prevalence of tetracycline and doxycycline resistance in Brucella was low (1.7%) but increased over time. This increase in tetracycline and doxycycline resistance highlights the need for further research to understand resistance mechanisms and develop more effective treatments.

Genotype diversity of brucellosis agents isolated from humans and animals in Greece based on whole-genome sequencing.

BACKGROUND: Brucellosis is a zoonotic disease whose causative agent, Brucella spp., is endemic in many countries of the Mediterranean basin, including Greece. Although the occurrence of brucellosis must be reported to the authorities, it is believed that the disease is under-reported in Greece, and knowledge about the genomic diversity of brucellae is lacking. METHODS: Thus, 44 Brucella isolates, primarily B. melitensis, collected between 1999 and 2009 from humans and small ruminants in Greece were subjected to whole genome sequencing using short-read technology. The raw reads and assembled genomes were used for in silico genotyping based on single nucleotide substitutions and alleles. Further, specific genomic regions encoding putative virulence genes were screened for characteristic nucleotide changes, which arose in different genotype lineages. RESULTS: In silico genotyping revealed that the isolates belonged to three of the known sublineages of the East Mediterranean genotype. In addition, a novel subgenotype was identified that was basal to the other East Mediterranean sublineages, comprising two Greek strains. The majority of the isolates can be assumed to be of endemic origin, as they were clustered with strains from the Western Balkans or Turkey, whereas one strain of human origin could be associated with travel to another endemic region, e.g. Portugal. Further, nucleotide substitutions in the housekeeping gene rpoB and virulence-associated genes were detected, which were characteristic of the different subgenotypes. One of the isolates originating from an aborted bovine foetus was identified as B. abortus vaccine strain RB51. CONCLUSION: The results demonstrate the existence of several distinct persistent Brucella sp. foci in Greece. To detect these and for tracing infection chains, extensive sampling initiatives are required.

Brucella melitensis: Divergence Among Indian Strains and Genetic Characterization of a Strain Isolated from Cattle.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01081-w.

Trans-species transmission of Brucellae among ruminants hampering brucellosis control efforts in Egypt.

AIMS: This study aimed to identify the genotypic fingerprinting of Brucella melitensis biovar 3 isolates from ruminants in Kafr El-Sheikh, Egypt, to compare with other peers globally and to highlight the epidemiology and potential causes of brucellosis control failure. METHODS AND RESULTS: A multilocus variable-number tandem-repeat analysis (MLVA 16) was carried out on 41 B. melitensis bv3 isolates, 31 from the preferential hosts (28 sheep and three goats) and 10 from atypical hosts (nine cattle and one buffalo), identified by bacteriological and molecular techniques. MLVA-16 analysis revealed 19 genotypes with nine as singletons. The most prevalent genotypes were M3_K.E (3,5,3,13,1,1,3,3,7,43,8,7,6,7,5,3), M13_K.E (3,5,3,13,1,1,3,3,7,43,8,5,8,7,7,3) and M5_K.E (3,5,3,13,1,1,3,3,7,43,8,4,8,7,11,3) circulating between different animal species. The B. melitensis isolation from aborted cows in farms that had never reared small ruminants indicates the likelihood of cow to cow B. melitensis transmission. Different genotypes of B. melitensis could be isolated from the same animal. The local geographic distribution of genotypes showed a very close genetic relatedness with genotypes reported outside the study area. Worldwide, our genotypes were mostly related to the Western Mediterranean lineage and less likely to the America's clonal lineage. CONCLUSION: There is a high genetic similarity of B. melitensis bv3 genotypes among different ruminant species, and the same animal could be infected with different genotypes. There is a high probability of spreading of B. melitensis among atypical hosts in the absence of the original hosts. The genetic relatedness of B. melitensis bv3 genotypes in the study area with other different geographic areas highlighted the national and international ruminants movement role as a potential factor for maintaining B. melitensis infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Further investigations are required to understand the impact of the presence of more than one genotype of B. melitensis in the same animal on the efficacy of brucellosis control strategies.

Evaluation of clinical, diagnostic features and therapeutic outcome of neurobrucellosis: a case series and review of literature.

AIM: In this study, we aimed to discuss the clinical features, laboratory findings, treatment and outcome of seven cases of neurobrucellosis from a tertiary care center and review the available global literature. MATERIALS AND METHODS: The diagnosis of neurobrucellosis was established using the following criteria in our setting: (1) signs and symptoms of neurological infection with examination of cerebrospinal fluid (CSF) revealing signs of meningitis, (2) isolation of Brucella spp. from blood and/or CSF and/or antibody titer ≥1:160 in serum using standard agglutination test (SAT) and/or the presence of anti-Brucella antibodies in CSF and/or detection of Brucella spp.-specific DNA from CSF using PCR. A literature search was performed to review previous cases of neurobrucellosis published worldwide during the last 30 years. RESULTS: The proportion of neurobrucellosis was 2.8% in our setting. Fever with headache and altered sensorium were the major presenting complaints. Brucella melitensis was isolated from blood culture in 6 patients. From the literature search, a total of 221 cases of neurobrucellosis were reviewed and analyzed. Meningitis (32.6%), loss of hearing (25.8%) and encephalitis (14.9%) were the most common clinical features. Involvement of cranial nerves, polyradiculopathy and paraplegia were the major complications found in patients with neurobrucellosis. CONCLUSIONS: Neurobrucellosis should always be considered in the differential diagnosis of befitting neurological, rheumatological, and neuropsychiatric presentations in endemic regions for brucellosis. To prevent morbidity and mortality associated with neurobrucellosis, a multimodal diagnostic approach is essential for early and accurate diagnosis and effective treatment.

The Flagellar Transcriptional Regulator FtcR Controls Brucella melitensis 16M Biofilm Formation via a betI-Mediated Pathway in Response to Hyperosmotic Stress.

The expression of flagellar proteins in Brucella species likely evolved through genetic transference from other microorganisms, and contributed to virulence, adaptability, and biofilm formation. Despite significant progress in defining the molecular mechanisms behind flagellar gene expression, the genetic program controlling biofilm formation remains unclear. The flagellar transcriptional factor (FtcR) is a master regulator of the flagellar system's expression, and is critical for B. melitensis 16M's flagellar biogenesis and virulence. Here, we demonstrate that FtcR mediates biofilm formation under hyperosmotic stress. Chromatin immunoprecipitation with next-generation sequencing for FtcR and RNA sequencing of ftcR-mutant and wild-type strains revealed a core set of FtcR target genes. We identified a novel FtcR-binding site in the promoter region of the osmotic-stress-response regulator gene betI, which is important for the survival of B. melitensis 16M under hyperosmotic stress. Strikingly, this site autoregulates its expression to benefit biofilm bacteria's survival under hyperosmotic stress. Moreover, biofilm reduction in ftcR mutants is independent of the flagellar target gene fliF. Collectively, our study provides new insights into the extent and functionality of flagellar-related transcriptional networks in biofilm formation, and presents phenotypic and evolutionary adaptations that alter the regulation of B. melitensis 16M to confer increased tolerance to hyperosmotic stress.

Abnormal liver findings as the first manifestation in a patient with brucellosis : Case report.

In January 2019, a 30-year-old woman admitted to our inpatient department presented with undulating fever, pain in several joints, and significantly elevated liver enzymes and lactate dehydrogenase. After extended examination, infection with Brucella melitensis with liver, musculoskeletal, and pulmonary involvement was diagnosed and treated. Diagnosis was based on clinical examination, laboratory findings including seroconversion as a proof of immune response, magnetic resonance imaging, three-phase bone scintigraphy, and F­18 FDG-PET (F-18 Flourdeoxyglucose positron emission tomography) illustrating the bone involvement and its normalization upon treatment. After treatment the patient showed a remarkable improvement of clinical symptoms within a short period. The patient remained symptom free and polymerase chain reaction (PCR) testing for brucellosis was negative, even at the follow-up examination 12 months after the end of the antibiotic therapy. The family members were also examined due to the similar travel history, and by this, brucellosis was also diagnosed in her husband but not in her children.

An Extracytoplasmic Function Sigma/Anti-Sigma Factor System Regulates Hypochlorous Acid Resistance and Impacts Expression of the Type IV Secretion System in Brucella melitensis.

The intracellular bacterial pathogen Brucella causes persistent infections in various mammalian species. To survive and replicate within macrophages, these bacteria must be able to withstand oxidative stresses and express the type IV secretion system (T4SS) to evade host immune responses. The extracytoplasmic function (ECF) sigma factor system is a major signal transduction mechanism in bacteria that senses environmental cues and responds by regulating gene expression. In this study, we defined an ECF σ bcrS and its cognate anti-σ factor abcS in Brucella melitensis M28 by conserved domain analysis and a protein interaction assay. BcrS directly activates an adjacent operon, bcrXQP, that encodes a methionine-rich peptide and a putative methionine sulfoxide reductase system, whereas AbcS is a negative regulator of bcrS and bcrXQP. The bcrS-abcS and bcrXQP operons can be induced by hypochlorous acid and contribute to hypochlorous acid resistance in vitro. Next, RNA sequencing analysis and genome-wide recognition sequence search identified the regulons of BcrS and AbcS. Interestingly, we found that BcrS positively influences T4SS expression in an AbcS-dependent manner and that AbcS also affects T4SS expression independently of BcrS. Last, we demonstrate that abcS is required for the maintenance of persistent infection, while bcrS is dispensable in a mouse infection model. Collectively, we conclude that BcrS and AbcS influence expression of multiple genes responsible for Brucella virulence traits. IMPORTANCEBrucella is a notorious intracellular pathogen that induces chronic infections in animals and humans. To survive and replicate within macrophages, these bacteria require a capacity to withstand oxidative stresses and to express the type IV secretion system (T4SS) to combat host immune responses. In this study, we characterized an extracytoplasmic function sigma/anti-sigma factor system that regulates resistance to reactive chlorine species and T4SS expression, thereby establishing a potential link between two crucial virulence traits of Brucella. Furthermore, the anti-sigma factor AbcS contributes to Brucella persistent infection of mice. Thus, this work provides novel insights into Brucella virulence regulation as well as a potential drug target for fighting Brucella infections.

Brucellosis Outbreak Traced to Commercially Sold Camel Milk through Whole-Genome Sequencing, Israel.

Brucellosis, a neglected zoonotic disease acquired from contaminated food products, remains a public health concern worldwide. We describe an outbreak in which commercially sold camel milk containing Brucella melitensis was distributed across Israel. Whole-genome sequencing linked patients infected with B. melitensis to wholesale camel milk and unregulated livestock trade.

Rickettsia burneti and Brucella melitensis co-infection: a case report and literature review.

Rickettsia is the pathogen of Q fever, Brucella ovis is the pathogen of brucellosis, and both of them are Gram-negative bacteria which are parasitic in cells. The mixed infection of rickettsia and Brucella ovis is rarely reported in clinic. Early diagnosis and treatment are of great significance to the treatment and prognosis of brucellosis and Q fever. Here, we report a case of co-infection Rickettsia burneti and Brucella melitensis. The patient is a 49-year-old sheepherder, who was hospitalized with left forearm trauma. Three days after admission, the patient developed fever of 39.0°C, accompanied by sweating, fatigue, poor appetite and headache. Indirect immunofluorescence (IFA) was used to detect Rickettsia burneti IgM. After 72 hours of blood culture incubation, bacterial growth was detected in aerobic bottles, Gram-negative bacilli were found in culture medium smear, the colony was identified as Brucella melitensis by mass spectrometry. Patients were treated with doxycycline (100 mg bid, po) and rifampicin (600 mg qd, po) for 4 weeks. After treatment, the symptoms disappeared quickly, and there was no sign of recurrence or chronic infection. Q fever and Brucella may exist in high-risk practitioners, so we should routinely detect these two pathogens to prevent missed diagnosis.

Isolation of Brucella melitensis biovar 1 from human milk confirms breastfeeding as a possible route for infant infection.

Brucellosis during pregnancy is associated with major concerns for mothers but, it is still not clear whether the infection could be transmitted through breastfeeding to newborns. This study aimed at evaluating the shedding of Brucella melitensis through human milk. We describe phenotypic and genotypic characterization of Brucella isolate from human milk. The characterized isolate by Bruce-ladder PCR, AMOS PCR and biotyping confirmed the presence of Brucella melitensis biovar 1 from human milk. However, the breastfeeding of newborn baby induced no serious abnormality, although occasional weakness, loss of appetite and vomiting were reported by the parents. All Brucella serological tests including RBT, SAT and 2 ME test were also positive for the baby and her mother, although the blood culture was negative for the baby. Evaluation of the blood DNA from mother and her baby also confirm the presence of Brucella melitensis in the blood samples. Therefore, the isolation of B. melitensis biovar 1 from human milk as well as presence of Brucella melitensis in the blood samples confirms breastfeeding as a possible route for infant infection.

Omp31 of Brucella Inhibits NF-κB p65 Signaling Pathway by Inducing Autophagy in BV-2 Microglia.

Neurobrucellosis is a serious central nervous system (CNS) inflammatory disorder caused by Brucella, and outer membrane protein-31 (Omp31) plays an important role in Brucella infection. This study aims to determine whether Omp31 can induce autophagy in BV-2 microglia. Another goal of the study is to further examine the effect of autophagy on the nuclear transcription factor κB (NF-κB) p65 signaling pathway. We observed that Omp31 stimulated autophagy by increasing microtubule-associated protein 1 light chain 3B (LC3B-II) levels and inducing autophagosome formation at 6 h and 12 h. Concomitantly, Omp31 induced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression in a time-dependent manner but reduced the expression of TNF-α at 6 h. We utilized Omp31 with or without rapamycin or 3-methyladenine (3-MA) to treat BV-2 microglia, and it demonstrated further that Omp31 induced autophagy by promoting LC3B-II, Beclin-1 proteins expression and inhibiting the p62 protein levels. Furthermore, we explored the effects of autophagy on the NF-κB p65 pathway through western blot analysis, RT-qPCR assay, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The data suggest that Omp31 as well as rapamycin, the autophagy inducer, can decrease TNF-α levels through the inhibition of the NF-κB p65 signaling pathway. Taken together, Omp31 can function as a catalyst in both autophagy induction and NF-κB p65 signal inhibition. Furthermore, Omp31-induced autophagy may inhibit the expression of TNF-α by negatively regulating NF-κB p65 signaling pathway.

Brucella melitensis invA gene (BME_RS01060) transcription is promoted under acidic stress conditions.

The invA gene of Brucella melitensis codes for a NUDIX (nucleoside diphosphate linked to moiety X) hydrolase related to invasiveness. The objective of this work was to evaluate invA transcription under acidic conditions. The invA gene transcription was up regulated at pH 3 and pH 5 observed with semiquantitative real-time PCR in B. melitensis 133 strain. Results indicated that invA gene transcription at pH 3 showed a basal and decreased transcription compared to that of pH 5 incubation. Transcription levels of the dnaK gene were similar to those obtained with invA gene. The survival rates of wild type and invA mutant strains at pH 5 were above 90% in all post-incubation times. In contrast, at pH 3 there was a time-dependent reduction on both strains at 15 min (P < 0.05). These results suggest that invA gene transcription is promoted under acidic conditions in Brucella melitensis.

A case report of endocarditis and spondylitis caused by Brucella melitensis biovar 3.

BACKGROUND: This case report describes the clinical process of a shepherd who suffered brucellosis-related endocarditis (BE) and spondylitis (BS) and was infected with Brucella melitensis biovar 3 (B. melitensis biovar 3). CASE PRESENTATION: A 55-year-old male patient was admitted to The First Affiliated Hospital of Shihezi University on October 11, 2018, due to over 3 months of intermittent fever, back pain, and heart trouble. The Rose Bengal Plate test was positive, the standard agglutination test titer for brucellosis was 1/800, and the blood culture was positive for B. melitensis biovar 3. Three instances of transthoracic echocardiography examination at days 1, 25, and 376 after admission to the hospital and magnetic resonance imaging (MRI) and computed tomography (CT) checks at days 5 and 38 revealed that the size of the vegetation on the posterior leaflet of the mitral valve increased from 0.7 × 1.4 cm to 1.2 × 1.5 cm and that the left atrium and ventricle were enlarged. The MRI and CT results showed hyperplasia of the second and third vertebra, a cold abscess formed on both sides of the psoas major muscles, and the vertebra hyperplasia became aggravated at a later time point. The patient's situation deteriorated, and heart failure was discovered on October 22, 2019. At the moment of submission of this manuscript, the patient remains in bed at home because of severe debility caused by brucellosis. CONCLUSIONS: This is the first reported case of endocarditis combined with spondylitis caused by B. melitensis biovar 3 in a shepherd. Brucellosis infection can cause work-power losses because of misdiagnosis or a lack of proper treatment. Early diagnosis and treatment are essential for a successful outcome.

Ile258Met mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase significantly enhances catalytic efficiency, cofactor affinity, and thermostability.

NAD(H)-dependent 7α-hydroxysteroid dehydrogenase catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. Here, we designed mutations of Ile258 adjacent to the catalytic pocket of Brucella melitensis 7α-hydroxysteroid dehydrogenase. The I258M variant gave a 4.7-fold higher kcat, but 4.5-fold lower KM, compared with the wild type, resulting in a 21.8-fold higher kcat/KM value for chenodeoxycholic acid oxidation. It presented a 2.0-fold lower KM value with NAD+, suggesting stronger binding to the cofactor. I258M produced 7-oxolithocholic acid in the highest yield of 92.3% in 2 h, whereas the wild-type gave 88.4% in 12 h. The I258M mutation increased the half-life from 20.8 to 31.1 h at 30 °C. Molecular dynamics simulations indicated increased interactions and a modified tunnel improved the catalytic efficiency, and enhanced rigidity at three regions around the ligand-binding pocket increased the enzyme thermostability. This is the first report about significantly improved catalytic efficiency, cofactor affinity, and enzyme thermostability through single site-mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase. KEY POINTS: • Sequence and structure analysis guided the site mutation design. • Thermostability, catalytic efficiency and 7-oxo-LCA production were determined. • MD simulation was performed to indicate the improvement by I258M mutation.

New Brucella variant isolated from Croatian cattle.

BACKGROUND: A novel Brucella strain closely related to Brucella (B.) melitensis biovar (bv) 3 was found in Croatian cattle during testing within a brucellosis eradication programme. CASE PRESENTATION: Standardised serological, brucellin skin test, bacteriological and molecular diagnostic screening for Brucella infection led to positive detection in one dairy cattle herd. Three isolates from that herd were identified to species level using the Bruce ladder method. Initially, two strains were typed as B. melitensis and one as B. abortus, but multiplex PCR based on IS711 and the Suis ladder showed that all of them to belong to B. melitensis, and the combination of whole-genome and multi-locus sequencing as well as Multi-Locus Variable numbers of tandem repeats Analysis (MLVA) highlighted a strong proximity within the phylogenetic branch of B. melitensis strains previously isolated from Croatia, Albania, Kosovo and Bosnia and Herzegovina. Two isolates were determined to be B. melitensis bv. 3, while the third showed a unique phylogenetic profile, growth profile on dyes and bacteriophage typing results. This isolate contained the 609-bp omp31 sequence, but not the 723-bp omp31 sequence present in the two isolates of B. melitensis bv. 3. CONCLUSIONS: Identification of a novel Brucella variant in this geographic region is predictable given the historic endemicity of brucellosis. The emergence of a new variant may reflect a combination of high prevalence among domestic ruminants and humans as well as weak eradication strategies. The zoonotic potential, reservoirs and transmission pathways of this and other Brucella variants should be explored.

Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO.

BACKGROUND: UTP-glucose-1-phosphoryl transferase (UGPase) catalyzes the synthesis of UDP-glucose, which is essential for generating the glycogen needed for the synthesis of bacterial lipopolysaccharide (LPS) and capsular polysaccharide, which play important roles in bacterial virulence. However, the molecular function of UGPase in Brucella is still unknown. RESULTS: In this study, the ubiquitination modification of host immune-related protein in cells infected with UGPase-deleted or wild-type Brucella was analyzed using ubiquitination proteomics technology. The ubiquitination modification level and type of NF-κB Essential Modulator (NEMO or Ikbkg), a molecule necessary for NF-κB signal activation, was evaluated using Coimmunoprecipitation, Western blot, and dual-Luciferase Assay. We found 80 ubiquitin proteins were upregulated and 203 ubiquitin proteins were downregulated in cells infected with B. melitensis 16 M compared with those of B. melitensis UGPase-deleted strain (16 M-UGPase-). Moreover, the ubiquitin-modified proteins were mostly enriched in the categories of regulation of kinase/NF-κB signaling and response to a bacterium, suggesting Brucella UGPase inhibits ubiquitin modification of related proteins in the host NF-κB signaling pathway. Further analysis showed that the ubiquitination levels of NEMO K63 (K63-Ub) and Met1 (Met1-Ub) were significantly increased in the 16 M-UGPase--infected cells compared with that of the 16 M-infected cells, further confirming that the ubiquitination levels of NF-κB signaling-related proteins were regulated by the bacterial UGPase. Besides, the expression level of IκBα was decreased, but the level of p-P65 was significantly increased in the 16 M-UGPase--infected cells compared with that of the 16 M- and mock-infected cells, demonstrating that B. melitensis UGPase can significantly inhibit the degradation of IκBα and the phosphorylation of p65, and thus suppressing the NF-κB pathway. CONCLUSIONS: The results of this study showed that Brucella melitensis UGPase inhibits the activation of NF-κB by modulating the ubiquitination of NEMO, which will provide a new scientific basis for the study of immune mechanisms induced by Brucella.

Immunization effect of lipopolysaccharide antigen in conjugation with PLGA nanoparticles as a nanovaccine against Brucella melitensis infection.

Brucella is an infectious disease with difficult treatment faced with drug resistance and recurrence of infection. Despite advances in the development of effective vaccines against brucellosis infections, there is still a need for more effective vaccine against brucellosis. In this study, we developed a nanovaccine for delivery of lipopolysaccharide Brucella melitensis antigen to the immune system using PLGA nanoparticles to prevent Brucella infection, which is associated with the stimulation of both humoral and cellular immune systems. In particular, we determined the rate of produced immunoglobulines and their functional effectiveness on the immune system by carring out opsonophagocytosis and challenge tests. According to the results, it was determined that PLGA improve the delivery of LPS antigen to the immune system to enhance the production of immunoglobulins levels and their efficiency to remove Brucella bacteria.