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Nano-based drug delivery systems are increasingly used for diagnosis, prevention and treatment of several diseases, thanks to several beneficial properties, including the ability to target specific cells or organs, allowing to reduce treatment costs and side effects frequently associated with chemotherapeutic medications, thereby improving treatment compliance of patients. In the field of communicable diseases, especially those caused by intracellular bacteria, the delivery of antibiotics targeting specific cells is of critical importance to maximize their treatment efficacy. Brucella melitensis, an intracellular obligate bacterium surviving and replicating inside macrophages is hard to be eradicated, mainly because of the low ability of antibiotics to enter these phagocityc cells . Although different antibiotics regimens including gentamicin, doxycycline and rifampicin are in fact used against the Brucellosis, no efficient treatment has been attained yet, due to the intracellular life of the respective pathogen. Nano-medicines responding to environmental stimuli allow to maximize drug delivery targeting macropages, thereby boosting treatment efficacy. Several drug delivery nano-technologies, including solid lipid nanoparticles, liposomes, chitosan, niosomes, and their combinations with chitosan sodium alginate can be employed in combination of antibiotics to successfully eradicate Brucellosis infection from patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Masson SAS.)

Brucella melitensis (B. melitensis) is an important facultative intracellular bacterium that causes global zoonotic diseases. Continuous intracellular survival and replication are the main obstruction responsible for the accessibility of prevention and treatment of brucellosis. Bacteria respond to complex environment by regulating gene expression. Many regulatory factors function at loci where RNA polymerase initiates messenger RNA synthesis. However, limited gene annotation is a current obstacle for the research on expression regulation in bacteria. To improve annotation and explore potential functional sites, we proposed a novel genome-wide method called Capping-seq for transcription start site (TSS) mapping in B. melitensis. This technique combines capture of capped primary transcripts with Single Molecule Real-Time (SMRT) sequencing technology. We identified 2,369 TSSs at single nucleotide resolution by Capping-seq. TSSs analysis of Brucella transcripts showed a preference of purine on the TSS positions. Our results revealed that -35 and -10 elements of promoter contained consensus sequences of TTGNNN and TATNNN, respectively. The 5' ends analysis showed that 57% genes are associated with more than one TSS and 47% genes contain long leader regions, suggested potential complex regulation at the 5' ends of genes in B. melitensis. Moreover, we identified 52 leaderless genes that are mainly involved in the metabolic processes. Overall, Capping-seq technology provides a unique solution for TSS determination in prokaryotes. Our findings develop a systematic insight into the primary transcriptome characterization of B. melitensis. This study represents a critical basis for investigating gene regulation and pathogenesis of Brucella., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Brucellosis is a neglected zoonotic disease of remarkable importance worldwide. The focus of this systematic review was to investigate occupational brucellosis and to identify the main infection risks for each group exposed to the pathogen. Seven databases were used to identify papers related to occupational brucellosis: CABI, Cochrane, Pubmed, Scielo, Science Direct, Scopus and Web of Science. The search resulted in 6123 studies, of which 63 were selected using the quality assessment tools guided from National Institutes of Health (NIH) and Case Report Guidelines (CARE). Five different job-related groups were considered greatly exposed to the disease: rural workers, abattoir workers, veterinarians and veterinary assistants, laboratory workers and hunters. The main risk factors and exposure sources involved in the occupational infection observed from the analysis of the articles were direct contact with animal fluids, failure to comply with the use of personal protective equipment, accidental exposure to live attenuated anti-brucellosis vaccines and non-compliance with biosafety standards. Brucella species frequently isolated from job-related infection were Brucella melitensis, Brucella abortus, Brucella suis and Brucella canis. In addition, a meta-analysis was performed using the case-control studies and demonstrated that animal breeders, laboratory workers and abattoir workers have 3.47 [95% confidence interval (CI); 1.47–8.19] times more chance to become infected with Brucella spp. than others individuals that have no contact with the possible sources of infection. This systematic review improved the understanding of the epidemiology of brucellosis as an occupational disease. Rural workers, abattoir workers, veterinarians, laboratory workers and hunters were the groups more exposed to occupational Brucella spp. infection. Moreover, it was observed that the lack of knowledge about brucellosis among frequently exposed professionals, in addition to some behaviors, such as negligence in the use of individual and collective protective measures, increases the probability of infection. Author summary: Brucellosis is a zoonotic bacterial infection of major importance worldwide, affecting not only domestic animals but different wildlife species. Due to its ways of transmission, direct or indirect contact with infected animals or their contaminated biological products, the disease exhibits a strong occupational character. This systematic review addressed the main occupations affected by Brucella spp. infection, due to the regular exposure to aerosol and contact of non-intact skin (e.g. wounds and abrasion) with infected materials, such as carcasses, viscera and live attenuated anti-brucellosis vaccines. The main risk factors for the disease were identified, as well as the most common forms of exposure to the pathogen. In addition, the most frequently Brucella species isolated from farmers, abattoir workers, veterinarians and veterinary technicians, laboratory workers and hunters were also described. The constant contact with the pathogen, the lack of information and instructions to occupational groups exposed, as well as the low adhesion to personal protective equipment in the work environment are determining factors for the occurrence of brucellosis among these individuals. [ABSTRACT FROM AUTHOR]

Introduction: The causes of femoral head necrosis mainly include trauma, corticosteroid, and alcohol, among which Brucella is highly unusual. Patients and Methods: This paper reported three rare cases of femoral head necrosis due to Brucella, summarizing and analyzing the epidemiological history, clinical manifestations, laboratory examinations, imaging findings, individualized therapy, and biopsy results of patients with this disease. A literature review related to brucellosis and femoral head necrosis was conducted. Results: All three patients had an epidemic history of brucellosis. They suffered from hip pain and limitation of movement for months, and femoral head necrosis was confirmed by magnetic resonance imaging (MRI). Pathological Giemsa staining or real-time polymerase chain reaction (real-time PCR) confirmed Brucella melitensis (B. melitensis) as the cause of osteonecrosis. Each patient received individualized therapy according to the degree of osteonecrosis, which was based on anti-brucellosis medications and hip puncture and irrigation decompression. They were followed up for more than two years and rehabilitated well. Conclusion: Brucella infection should always be considered the cause of femoral head necrosis for patients with hip pain, especially for those with an epidemic history; early diagnosis and individual stepwise therapy can prevent the progression of osteonecrosis. [ABSTRACT FROM AUTHOR]

Brucellosis is a worldwide zoonosis caused by bacteria from the genus Brucella. Once established, it is very hard to eradicate this disease, since it contaminates animals, the environment, and humans, causing problems for veterinary and public health as well as wildlife protection programs. Swabs are used for sampling in bacteriological and/or molecular diagnostics, from seropositive animals with disease symptoms, from genitalia or tissue lesions, as well as from contaminated environments. The aim of this study was to compare main of the commercially used swab types for sampling and diagnostics of Brucella spp. and determine the optimal storage conditions and time frame for testing. To achieve this, we tested bacterial and molecular methods for detection of Brucella abortus, Brucella melitensis, and Brucella suis using nine swab types, all with different tip materials, treated immediately after spiking, after 72 h at +4°C, and after 72 h at -20°C. Flocked swabs showed the highest capacity to preserve bacterial viability and DNA quality, regardless the storage conditions. Flocked swabs immersed in a protective medium provided the best conditions for Brucella survival in all three storage conditions. At the same time, the efficacy of quantitative PCR (qPCR) detection for all swabs, including the positive control, was above 50%, irrespective of the storage conditions, while bacterial survival was significantly lowered when swabs were kept at +4°C or -20°C for 72 h (48.2% and 27.5%, respectively). Compared to the positive control and other types, the flocked swabs maintained higher reproducibility regarding their capacity to preserve live bacteria in all three storage conditions. IMPORTANCE In order to protect public and veterinary health from highly zoonotic bacteria such as members of the genus Brucella and prevent their dissemination into the environment, direct diagnostics are of utmost importance. However, in addition to the highly specific diagnostic tests, the sampling methods, time necessary for specimens to reach the laboratories, and transport conditions are important factors to consider in order to increase the sensitivity of performed tests, especially bacterial culturing and qPCR. This paper shows how different swab types and storage conditions influence classical bacteriological diagnostics of the most prevalent Brucella species - B. melitensis, B. abortus, and B. suis - but have little impact on molecular methods. The presented results highlight (i) the choice of swab regarding the storage and transport conditions, (ii) the importance of immediate swab treatment upon sampling, and (iii) that molecular methods do not depend on storage conditions, unlike classical bacteriological isolation.

Background: A new candidate vector vaccine against human brucellosis based on recombinant influenza viral vectors (rIVV) subtypes H5N1 expressing Brucella outer membrane protein (Omp) 16, L7/L12, Omp19 or Cu-Zn SOD proteins has been developed. This paper presents the results of the study of protection of the vaccine using on guinea pigs, including various options of administering, dose and frequency. Provided data of the novel vaccine candidate will contribute to its further movement into the preclinical stage study., Methods: General states of guinea pigs was assessed based on behavior and dynamics of a guinea pig weight-gain test. The effectiveness of the new anti-brucellosis vector vaccine was determined by studying its protective effect after conjunctival, intranasal and sublingual administration in doses 10 5 EID 50 , 10 6 EID 50 and 10 7 EID 50 during prime and boost vaccinations of animals, followed by challenge with a virulent strain of B. melitensis 16 M infection. For sake of comparison, the commercial B. melitensis Rev.1 vaccine was used as a control. The protective properties of vaccines were assessed by quantitation of Brucella colonization in organs and tissues of infected animals and compared to the control groups., Results: It was observed a gradual increase in body weight of guinea pigs after prime and booster immunization with the vaccine using conjunctival, intranasal and sublingual routes of administration, as well as after using various doses of vaccine. The most optimal way of using the vaccine has been established: double intranasal immunization of guinea pigs at a dose of 10 6 EID 50 , which provides 80% protection of guinea pigs from B. melitensis 16 M infection (P < 0.05), which is comparable to the results of the effectiveness of the commercial B. melitensis Rev.1 vaccine., Conclusions: We developed effective human vaccine candidate against brucellosis and developed its immunization protocol in guinea pig model. We believe that because of these studies, the proposed vaccine has achieved the best level of protection, which in turn provides a basis for its further promotion.

Bacterial signal transduction pathways are important for a variety of adaptive responses to environment, such as two-component systems (TCSs). In this paper, we reported the characterization of a transcriptional regulator in Azorhizobium caulinodans ORS571, ActR, with an N-terminal receiver domain and one C-terminal OmpR/PhoB-type DNA binding domain. Sequence analysis showed that ActR shared a high similarity with FtcR regulator of Brucella melitensis 16M known to be involved in flagellar regulation. The structural gene of this regulator was largely distributed in Alphaproteobacteria, in particular in Rhizobiales and Rhodobacterales, and was located within clusters of genes related to motility functions. Furthermore, we studied the biological function of ActR in A. caulinodans grown at the free-living state or in association with Sesbania rostrata by constructing actR gene deletion mutant. In the free-living state, the bacterial flagellum and motility ability were entirely deleted, the expression of flagellar genes was downregulated; and the exopolysaccharide production, biofilm formation, and cell flocculation decreased significantly compared with those of the wild-type strain. In the symbiotic state, Δ actR mutant strain showed weakly competitive colonization and nodulation on the host plant. These results illustrated that FtcR-like regulator in A. caulinodans is involved in flagellar biosynthesis and provide bacteria with an effective competitive nodulation for symbiosis. These findings improved our knowledge of FtcR-like transcriptional regulator in A. caulinodans. [ABSTRACT FROM AUTHOR]

Brucellosis is a worldwide bacterial zoonosis disease. Live attenuated Brucella vaccines have several drawbacks. Thus development of a safe and effective vaccine for brucellosis is a concern of many scientists. FliC protein contributes in virulence of Brucella; hence, it is a promising target for brucellosis vaccine. In this study, Mannosylated Chitosan Nanoparticles (MCN) loaded with FliC protein were synthesized as a targeted vaccine delivery system. The immunogenicity and protective efficacy of FliC and FliC-MCN against Brucella infection were evaluated in BALB/c mice. After cloning, expression and purification, FliC protein was loaded on MCN. The particle size, loading efficiency and in vitro release of the NPs were determined. Our investigation revealed that FliC and FliC-MCN could significantly increase specific IgG response (higher IgG2a titers). Besides, spleen cells from immunized mice produced high level of IFN-γ and IL-2 and low level IL-10 cytokines. Immunization with FliC and FliC-MCN conferred significant degree of protection against B. melitensis 16 M and B. abortus 544 infections. Overall these results indicate that FliC protein would be a novel potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus. Moreover, MCN could be used as an adjuvant and targeted vaccine delivery system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Brucellar spondylodiscitis, the most prevalent and significant osteoarticular presentation of human Brucellosis, is difficult to diagnose and usually yields irreversible neurologic deficits and spinal deformities. However, no animal models of Brucellar spondylodiscitis exist, allowing for preclinical investigations. The present study investigated whether intraosseous injection of attenuated Brucella melitensis vaccine into rabbits' lumbar vertebrae imitates the radiographic and histopathological characteristics of human Brucellar spondylodiscitis. Radiographic and histopathological analyses at 8 weeks postoperatively revealed radiographic changes within vertebral bodies and intervertebral discs, abscesses formation within the paravertebral soft tissue, and typical prominent inflammation response without caseous necrosis, which were largely comparable to human Brucellar spondylodiscitis. Such a medium-sized, surgically feasible rabbit model provides a promising in vivo setting for further preclinical investigation of Brucellar spondylodiscitis., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2019 Xiaoyu Cai et al.)

Brucellosis is a neglected zoonotic disease affecting livestock and humans that remains endemic in Ethiopia. Despite its prevalence, only a few studies have identified Brucella species circulating in livestock in the country. This study aimed to determine the Brucella species responsible for infections in livestock in the Afar region of Ethiopia and characterize the isolates using whole-genome single nucleotide polymorphism (wgSNP) analysis and in silico multi-locus sequence typing (MLST). Comparisons were made between Ethiopian Brucella and regional and global isolates to determine their phylogenetic relationships. Surveys conducted in May and October–November 2022 in six villages of the Amibara district involved the collection of vaginal swabs (n = 231) and milk samples (n = 17) from 32 sheep and 199 goats kept by 143 pastoral households reporting recent abortions in the animals. Brucella melitensis was detected in three sheep and 32 goats, i.e., 15% (35/231) of animals across 20% (29/143) of households using bacterial culture and PCR-based methods (bcsp31, AMOS, and Bruce-ladder multiplex PCR). Of the 35 positive animals, B. melitensis was isolated from 24 swabs, while the remaining 11 were culture-negative and detected only by PCR. The genomic DNA of the 24 isolates was sequenced using Illumina Novaseq 6000 and assembled using the SPAdes pipeline. Nine- and 21-locus MLST identified 23 isolates as genotype ST12, while one isolate could not be typed. The wgSNP-based phylogenetic analysis revealed that the Ethiopian isolates clustered within the African clade and were closely related to isolates from Somalia. Several virulence factors responsible for adhesion, intracellular survival, and regulatory functions were detected in all isolates. No antimicrobial resistance genes associated with resistance to drugs commonly used for treating brucellosis were detected. Since B. melitensis is prevalent in sheep and goats, vaccination with the B. melitensis Rev-1 vaccine is the recommended strategy in these pastoral systems to protect animal and human health. [ABSTRACT FROM AUTHOR]

In this study, the isolation, biotyping and molecular characterisation of Brucella melitensis from cattle, sheep and goats in North Cyprus are reported on. A total of 319 raw milk samples obtained from seropositive dairy livestock (190 cattle, 74 sheep and 55 goats) and tissue samples including the liver, spleen and abomasal contents obtained from 32 aborted foetal samples (5 cattle, 18 sheep and 9 goats) were analysed for the presence and characterisation of the agent. B. melitensis was isolated and identified from 90 out of 319 (28.2%) milk and 19 out of 32 (59.4%) foetal samples by conventional bacteriological methods. Identification of all 109 isolates was confirmed by using real-time PCR with genus and species-specific primers. Following the preliminary identification, 27 selected isolates representing various counties and herds were further analysed by conventional methods. Twenty (74.1%) isolates were identified as B. melitensis biovar 1 and seven (25.9%) were identified as B. melitensis biovar 3. The Bruce-ladder multiplex PCR assay revealed that all the isolates were field strains. The results of the present study confirmed the presence of B. melitensis in livestock including the cattle population in North Cyprus. Even though the majority of the samples came from seropositive cattle, Brucella abortus was not isolated in the study. The results also revealed the potential public health risk of brucellosis in livestock emphasising the need of implementing strict control and eradication strategies against the disease in animal populations in order to protect human health [ABSTRACT FROM AUTHOR]

Wildlife reservoirs of infectious diseases raise major management issues. In Europe, brucellosis has been eradicated in domestic ruminants from most countries and wild ruminants have not been considered important reservoirs so far. However, a high prevalence of Brucella melitensis infection has been recently identified in a French population of Alpine ibex (Capra ibex), after the emergence of brucellosis was confirmed in a dairy cattle farm and two human cases. This situation raised the need to identify the factors driving the persistence of Brucella infection at high prevalence levels in this ibex population. In the present paper, we studied the shedding pattern of B. melitensis in ibex fromBargyMassif, French Alps. Bacteriological examinations (1-15 tissues/samples per individual) were performed on 88 seropositive, supposedly infected and euthanized individuals. Among them, 51 (58%) showed at least one positive culture, including 45 ibex with at least one Brucella isolation from a urogenital sample or a lymph node in the pelvic area (active infection in organs in the pelvic area). Among these 45 ibex, 26 (30% of the total number of necropsied animals) showed at least one positive culture for a urogenital organ and were considered as being at risk of shedding the bacteria at the time of capture. We observed significant heterogeneity between sex-and-age classes: seropositive females were most at risk to excrete Brucella before the age of 5 years, possibly corresponding to abortion during the first pregnancy following infection such as reported in the domestic ruminants. The high shedding potential observed in young femalesmay have contributed to the self-sustainedmaintenance of infection in this population, whereas males are supposed to play a role of transmission between spatial units through venereal transmission during mating. This heterogeneity in the shedding potential of seropositive individuals should be considered in the future to better evaluate management scenarios in this system as well as in others. [ABSTRACT FROM AUTHOR]

Brucellosis is a zoonotic disease that can result in symptoms including infertility, abortion, testicular inflammation, and arthritis in affected sheep and cattle. The disease can spread through a range of mechanisms, making outbreaks difficult to control such that affected farms often suffer severe economic losses. In addition, humans can be affected by brucellosis, and the number of cases continues to rise annually. As rates of brucellosis in Xinjiang, China have been increasing substantially in recent years, this study was developed to assess brucellosis seroprevalence among herds of cattle, sheep, and camels in Xinjiang through the use of common diagnostic procedures like the Rose Bengal test (RBT) and PCR. A cross-sectional approach was used to analyze the seroprevalence of brucellosis across 720 total farm animals (320 camels, 250 sheep, and 150 cattle). RBT incidence rate for brucellosis was detected in 60 serum samples, 14 (4.38%) from camels, 45 (18%) from sheep, and 1 (0.67%) from cattle. PCR was performed as a confirmatory approach for these RBT-positive samples, with 55 ultimately being confirmed to be positive 13 (4.06%), 41 (16.4%), and 1 (0.67%) samples from camels, sheep, and cattle, respectively. In this survey, sheep exhibited the highest brucellosis seroprevalence using these two analytical techniques, while cattle exhibited the lowest seroprevalence and camels exhibited an intermediate rate. AMOS-PCR analyses identified Brucella melitensis as the unknown bacterium in some of these samples. These results offer new insight regarding brucellosis seroprevalence among farm animals that can be used to formulate more appropriate prevention and control policies, while also improving awareness of epidemic prevention efforts, the need for routine quarantine and disinfection, the benefits of scientific breeding and management, and approaches to improving breeding efficiency for the camel, cattle, and sheep industries. [ABSTRACT FROM AUTHOR]

A retrospective study was conducted in Abergele and Ziquala districts in Ethiopia to investigate the occurrence, impact, and potential risk factors for abortion in small ruminants linked to a regional abortion storm. Affected (case) and nonaffected (control) villages were compared to assess infectious and noninfectious risk factors causing abortions. A case village was defined as a village with abortion seen in all households enrolled in the study, while a control village is characterized by presence of abortion in two and fewer households. A questionnaire survey, focus group discussions, and serological examinations were used to assess the differences in the abortion rate between the case and control villages. The Rose Bengal Plate Test for Brucellosis, Toxoplasma gondii Antibody Test Kit for Toxoplasma gondii, and ELISA for Chlamydophila abortus and Coxiella burnetii were used to detect antibodies. Per village 15 household flocks were selected. In the case villages, all flocks were affected by abortion (a mean abortion of 13 animals in Abergele and 9 in Ziquala). In contrast, only three households reported abortions in the control villages. A total of 176 blood samples were collected from the case and control villages for further laboratory diagnosis of possible causes of abortion. Of the examined flocks, 17%, 10.2%, and 2.8% were positive for Coxiella burnetii, Toxoplasma gondii, and Chlamydophila abortus, respectively. However, antibodies against Brucella melitensis were not detected. While the seroprevalence was greater for some infectious agents of abortion, there was no significant difference between the case and control villages. It is clear that the abortion problem in the study villages is complex and likely due to a mix of poor husbandry practices and the presence of infectious diseases. To better understand the underlying causes of abortion, there is a need to conduct a longitudinal study involving testing for more pathogens at the household level combined with reliable data on husbandry practices. [ABSTRACT FROM AUTHOR]

Brucellosis is a chronic and debilitating disease in humans, causing great economic losses in the livestock industry. Making an effective vaccine is one of the most important concerns for this disease. The new mRNA vaccine technology due to its accuracy and high efficiency has given promising results in various diseases. The objective of this research was to create a novel mRNA vaccine with multiple epitopes targeting Brucella melitensis. Seventeen antigenic proteins and their appropriate epitopes were selected with immunoinformatic tools and surveyed in terms of toxicity, allergenicity, and homology. Then, their presentation and identification by MHC cells and other immune cells were checked with valid tools such as molecular docking, and a multi-epitope protein was modeled, and after optimization, mRNA was analyzed in terms of structure and stability. Ultimately, the immune system's reaction to this novel vaccine was evaluated and the results disclosed that the designed mRNA construct can be an effective and promising vaccine that requires laboratory and clinical trials. [ABSTRACT FROM AUTHOR]

Brucella spp. are facultative intracellular pathogens that cause zoonosis- brucellosis worldwide. There has been a trend of the re-emergence of brucellosis worldwide in recent years. The epidemic situation of brucellosis is serious in Xinjiang. To analyze the epidemic situation of Brucella spp. in Xinjiang among humans and animals, this study identified 144 Brucella isolates from Xinjiang using classical identification and 16 S rRNA sequencing. MLVA, drug resistance testing, and wgSNP detection were also performed. At the same time, analysis was conducted based on the published data of Brucella isolates worldwide. The results showed that the dominant species was B. melitensis biovar 3, which belonged to GT42 (MLVA-8 typing) and the East Mediterranean lineage. The correlation among isolates was high both in humans or animals. The isolates in Xinjiang exhibited higher polymorphism compared to other locations in China, with polymorphism increasing each year since 2010. No amikacin/kanamycin-resistant strains were detected, but six rifampicin-intermediate isolates were identified without rpoB gene variation. The NJ tree of the wgSNP results indicated that there were three main complexes of the B. melitensis epidemic in Xinjiang. Based on the results of this study, the prevention and control of brucellosis in Xinjiang should focus on B. melitensis, particularly strains belonging to B. melitensis bv.3 GT42 (MLVA-8 typing) and East Mediterranean lineage. Additionally, the rifampicin- and trimethoprim-sulfamethoxazole- resistance of isolates in Xinjiang should be closely monitored to avoid compromising the therapeutic efficacy and causing greater losses. These results provide essential data for the prevention and control of brucellosis in Xinjiang and China. Although the isolates from Xinjiang have significant characteristics among Chinese isolates and can reflect the epidemiological situation of brucellosis in China to some extent, this study cannot represent the characteristics of isolates from other regions. [ABSTRACT FROM AUTHOR]

Although liver involvement is frequently seen in brucellosis, acute hepatitis is a rare clinical entity. In its progress, haematological findings are non-specific and vary in respect to severity. In this paper, we present a case of brucellosis with acute hepatitis and bicytopenia without anaemia. A 19-year-old man presented with a 2-week history of fever, sweating, low back and leg pain, lassitude, loss appetite, nausea and vomiting. He gave a history of raw milk ingestion and animal contact. Physical examination showed signs of icteric skin and sclera, tenderness in the right hypochondriac region and hepatosplenomegaly. On admission to hospital, laboratory tests showed WBC 3500/mmc (polymorphs 63% and lymphocytes 33%), haemoglobin 13.8 g/dL, platelet 89000/mmc, erythrocyte sedimentation rate 19 mm/h, and C-reactive protein 21.7 mg/dL (N<0.8 mg/dL). Biochemical tests were as follows: AST 771 U/L, ALT 471 U/L, ALP 355 U/L, GGT 432 U/L, total bilirubin 2.61 mg/dL, direct bilirubin 1.45 mg/dL and albumin 3.7 g/dL. Viral hepatitis markers were found to be negative (HBsAg, anti-HBc total, anti-HBc IgM, anti-HAV IgM, and anti-HCV). Blood culture grew Brucella melitensis. Leukopenia and thrombocytopenia returned to normal levels at the 7th and 14th day of his admission, respectively. Liver function tests improved at the 28th day. Treatment of the brucellosis was performed with antibiotics (tetracycline 500 mg orally four times daily for 6 weeks and streptomycin 1 g IM once daily for 21 days). Finally, a case of brucellosis with acute hepatitis and bicytopenia was treated with a successful outcome. In conclusion, we suggest that due consideration be taken of bicytopenia/pancytopenia and acute hepatitis in brucellosis cases in Turkey, an endemic region.

Brucella melitensis is a major livestock bacterial pathogen and zoonosis, causing disease and infection-related abortions in small ruminants and humans. A considerable burden to animal-based economies today, the presence of Brucella in Neolithic pastoral communities has been hypothesised but we lack direct genomic evidence thus far. We report a 3.45X B. melitensis genome preserved in an ~8000 year old sheep specimen from Menteşe Höyük, Northwest Türkiye, demonstrating that the pathogen had evolved and was circulating in Neolithic livestock. The genome is basal with respect to all known B. melitensis and allows the calibration of the B. melitensis speciation time from the primarily cattle-infecting B. abortus to approximately 9800 years Before Present (BP), coinciding with a period of consolidation and dispersal of livestock economies. We use the basal genome to timestamp evolutionary events in B. melitensis, including pseudogenization events linked to erythritol response, the supposed determinant of the pathogen's placental tropism in goats and sheep. Our data suggest that the development of herd management and multi-species livestock economies in the 11th–9th millennium BP drove speciation and host adaptation of this zoonotic pathogen. Brucella melitensis is a zoonotic bacterial pathogen of livestock that can infect humans and causes brucellosis. Here, the authors sequence an ancient specimen of B. melitensis and show that the species emerged in the Neolithic period, around the time of development of animal management practices. [ABSTRACT FROM AUTHOR]

This study was carried out to evaluate Brucella spp. isolated from various tissue samples of aborted sheep and bovine fetuses sent to the laboratory of Department of Microbiology, Faculty of Veterinary Medicine, Kafkas University between 2011 and 2023 years and determine the Brucella species and biotype diversity that carry a higher risk for abortion complications in these animals. In this context, 155 Brucella spp. isolates obtained from aborted fetuses were identified by species-specific Bruceladder PCR and biotyped using conventional biotyping methods. As a result of the study, B. melitensis and B. abortus were identified in 92.5% (n=74) and 7.5% (n=6) of sheep, B. abortus and B. melitensis were identified in 80% (n=60) and 20% (n=15) of cattle, respectively. B. melitensis biotype 2 in sheep and B. abortus biotype 3 in cattle were found as the dominant biotypes in these definitive hosts. In the Kars region, where brucellosis is endemic, while the biotype responsible for cattle brucellosis (B. abortus biotype 3) maintained its dominance over a 20-year period, there is a profile change from B. melitensis biotype 3 to B. melitensis biotype 2 in sheep. Considering the period covered by the study and the sample size analyzed, the data obtained provide up-to-date and important information about Brucella species and biotypes in Kars region and the animal species that host these agents. [ABSTRACT FROM AUTHOR]

Brucellosis is a globally zoonotic bacterial disease of humans and various animals including goats, sheep, and cattle. Brucella melitensis M5-90, a live attenuated vaccine strain, has been widely used to prevent brucellosis in goats and sheep. However, the molecular mechanisms governing protective immunity response in non-professional phagocytes infected with B. melitensis M5-90 have not been fully investigated, especially in goats. In our research, goat fibroblasts were used as in vitro models to determine these mechanisms by transcriptome analysis. After incubating with B. melitensis M5-90 3 h, the infected goat fibroblasts were collected at 0 h, 4 h, 24 h, 48 h and 72 h for RNA-seq. The results indicated that there were totally 11,819 differentially expressed genes (DEGs) and 777 differentially expressed (DE) miRNAs found in experiment groups compared with the control groups (|log2 (Foldchange)|=1, FDR<0.05). GO and KEGG enrichment analyses revealed that down-regulated genes were involved in the riboflavin metabolism and positive regulation of IL-8 secretion pathway. The up-regulated genes were mainly involved in adaptive immunity, including TNF signaling pathway, MAPK signaling pathway and JAK/STAT pathway. Additionally, cytokine-cytokine receptor interaction, natural killer cell mediated cytotoxicity and toll-like receptor signaling pathway, which associated with innate immunity pathways, were also induced. Based on the Pearson correlation coefficients and prediction results of TargetScan and miRanda, the miRNA-mRNA networks of NFKB1, IFNAR2 and IL10RB were constructed and verified in goat fibroblasts by qPCR, which demonstrated that goat fibroblasts displayed immunomodulatory properties. Our findings provide a deeper insight into the host miRNA-driven B. melitensis defense mechanism and reveal the transcriptome changes involved in the innate and adaptive immune response of the goats to B. melitensis infection. [ABSTRACT FROM AUTHOR]

The control and eradication of brucellosis represents a critical objective for Veterinary and Health Authorities across several countries globally. Efficient surveillance programs play a pivotal role in detecting and managing outbreaks. Epidemiological investigations significantly benefit from standardized and efficient molecular typing techniques and analytical tools, enabling public health laboratories to identify the origin of outbreaks. This study aimed to sequence Brucella spp. strains isolated in Iraq from different ruminant species to verify their molecular epidemiological correlations and, above all, to shed a light on how these Iraqi isolates are positioned in the phylogenetic context of Brucella spp. The 35 isolates under study were from abortion, milk, placenta, and the fetal membranes of sheep, cattle, and buffalo. Genotyping involved various techniques: MLVA-16, Whole Genome Sequencing, MLST, and cgMLST. All the Iraqi isolates from our study clustered in MLVA-16 within the East Mediterranean clade, and all but one grouped together in the same branch of the MST tree. MST analysis showed the minimum distance of one allele between the studied isolates, except for one strain from buffalo, which was positioned farther away from the rest of the isolates. In cgMLST, the majority of strains grouped within a large cluster predominantly comprising genotypes from the Middle East. The application of different control measures in different territories based on molecular epidemiological studies would increase the chances of maximizing public health benefits and minimizing the spread of infection to disease-free or lower prevalence areas. [ABSTRACT FROM AUTHOR]

Background: Spontaneous miscarriage, a leading health concern globally, often occurs due to various factors, including infections. Among these, Coxiella burnetii and Brucella spp. may have adverse effects on pregnancy outcomes. While previous research has established a link between infections and spontaneous miscarriage, our study aimed specifically to investigate the presence of these two pathogens in abortion samples from women who experienced spontaneous miscarriages in Iran. Our study can add to the existing knowledge by focusing on Iran, a region with a high prevalence of C. burnetii and Brucella spp. As a result, it could provide a better understanding and unique insights into the relationship of these pathogens with spontaneous miscarriages in endemic regions. Methods: From March 2021 to March 2022, a total of 728 abortion samples (including placenta and cotyledon) were collected from 409 women who had experienced spontaneous miscarriages in the provinces of Tehran, Fars, and West Azerbaijan in Iran. The specimens included 467 Formalin-Fixed Paraffin-Embedded (FFPE) and 261 fresh frozen samples. After DNA extraction from abortion samples, the quantitative real-time PCR (qPCR) assay targeted a specific fragment of the IS1111 and IS711 elements for molecular identification of C. burnetii and Brucella spp., respectively. Furthermore, the qPCR assay employing specific primers for different species was used to determine the species of Brucella. Results: Among the studied women, 1 out of 409 (0.24%) samples tested positive for Brucella spp., specifically Brucella melitensis. There were no positive specimens for C. burnetii. Conclusions: Our study contributes to understanding the potential involvement of Brucella species in spontaneous infectious abortion within endemic regions. The identification of B. melitensis in this study highlights the need for further research in this area. However, while our results suggest a relatively low or zero identification of these pathogens in our sample population, this does not rule out the possibility of undetected infections. Therefore, it is critical to acknowledge the limitations of the molecular techniques used (qPCR), which may have potential limitations such as sensitivity and specificity. Moreover, because 64.15% of our samples were FFPE, the sensitivity of the qPCR test may be reduced. These raise concerns about the accuracy of the reported prevalence rates and the potential for false positives or negatives. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Human brucellosis, otherwise known as undulant fever, is one of the most widespread zoonotic diseases in the world. Even though 9%–15% of stillbirths are known to be caused by infectious diseases, the study of the link between human Brucella melitensis and the termination of births in humans is a topic that has received little attention. This study examines if there was an association between infection of undulant fever, an endemic zoonotic disease in the Maltese Islands from 1919 until 1954, and reproductive loss through stillbirths. Methods A univariate descriptive analysis was used to show the temporal trend of undulant fever time, as well as the age and sex distribution. Time series analysis was used to assess the relationship between time (months) and undulant fever cases with stillbirth proportions. Results On the island of Gozo, the majority of undulant fever cases for both males and females occurred in their reproductive period between 15 and 45 years of age. Based on regression analysis, undulant fever had a statistically significant effect on the stillbirth rate for males (t = 2.8986, p = 0.0039). The effect of undulant fever on stillbirths was not significant for females (p = 0.9103). Conclusion This paper highlights the importance of undulant fever as having implications for the health burden in pregnant women and potential fetal loss through stillbirths in the contemporary context.

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Background: Globalization and subsequent growth in international trade in animals and animal products has increased the importance of international disease reporting. Efficient and reliable surveillance systems are needed in order to document the disease status of a population at a given time. In this context, passive surveillance plays an important role in early warning systems. However, it is not yet routinely integrated in the assessment of disease surveillance systems because different factors like the disease awareness (DA) of people reporting suspect cases influence the detection performance of passive surveillance. In this paper, we used scenario tree methodology in order to evaluate and compare the quality and benefit of abortion testing (ABT) for Brucella melitensis (Bm) between the disease free situation in Switzerland (CH) and a hypothetical disease free situation in Bosnia and Herzegovina (BH), taking into account DA levels assumed for the current endemic situation in BH., Results: The structure and input parameters of the scenario tree were identical for CH and BH with the exception of population data in small ruminants and the DA in farmers and veterinarians. The sensitivity analysis of the stochastic scenario tree model showed that the small ruminant population structure and the DA of farmers were important influential parameters with regard to the unit sensitivity of ABT in both CH and BH. The DA of both farmers and veterinarians was assumed to be higher in BH than in CH due to the current endemic situation in BH. Although the same DA cannot necessarily be assumed for the modelled hypothetical disease free situation as for the actual endemic situation, it shows the importance of the higher vigilance of people reporting suspect cases on the probability that an average unit processed in the ABT-component would test positive., Conclusion: The actual sensitivity of passive surveillance approaches heavily depends on the context in which they are applied. Scenario tree modelling allows for the evaluation of such passive surveillance system components under assumed disease free situation. Despite data gaps, this is a real opportunity to compare different situations and to explore consequences of changes that could be made.

This paper describes a case of fibrinopurulent epididymitis and orchitis in a Merino ram owing to Brucella melitensis biovar 3, detected by histopathology, bacteriology, and PCR. Gross lesions included enlargement of the scrotum by abundant fibrinous exudate and adhesions between testicular tunics. Histopathologic lesions were characterized by epididymal abscesses and testicular atrophy. B. melitensis was cultured from the exudate as well as the internal organs of the ram. After conventional biotyping procedures were applied, the isolate was identified as B. melitensis biovar 3. The isolate was also confirmed as Brucella spp. by genus-specific PCR. [ABSTRACT FROM AUTHOR]

Brucellosis is a zoonotic infectious disease, which mainly present with lymphoreticular system invovement. However any organ system can be attacked by the microorganism. In this paper we present a 52-year-old female patient who was admitted to the Infectious Diseases Department with complaints of fatigue, arthralgias, fever, and weight loss. In the medical examination and radiological analysis bilateral pleural effusions and hepatosplenomegaly were detected. Serum transaminase levels were two times higher than the upper limits of normal. Abdominal ultrasound revealed sludge in the gallbladder and multiple hypodense splenic lesions (the largest was 1 cm in diameter). Brucella melitensis was isolated from the blood culture of the patient. Rifampicin (600mg/day) and doxycycline (200 mg/day) therapy was started. Follow-up chest radiography and ultrasonography revealed the absence of pleural effusion. Splenic lesions and hepatosplenomegaly were totally regressed. The patient has been followed for 3 months after 6 week antibiotic regimen without recurrence. Brucellosis was expected to be the cause of all pathological signs. [ABSTRACT FROM AUTHOR]

A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced B. melitensis DNA fragment.

The gene transfer agent (GTA) of the a-proteobacterium Rhodobacter capsulatus is a cell-controlled genetic exchange vector. Genes that encode the GTA structure are clustered in a 15-kb region of the R. capsulatus chromosome, and some of these genes show sequence similarity to known bacteriophage head and tail genes. However, the production of GTA is controlled at the level of transcription by a cellular two-component signal transduction system. This paper describes homologues of both the GTA structural gene cluster and the GTA regulatory genes in the a-proteobacteria Rhodopseudomonas palustris, Rhodobacter sphaeroides, Caulobacter crescentus, Agrobacterium tumefaciens and Brucella melitensis. These sequences were used in a phylogenetic tree approach to examine the evolutionary relationships of selected GTA proteins to these homologues and (pro)phage proteins, which was compared to a 16S rRNA tree. The data indicate that a GTA-like element was present in a single progenitor of the extant species that contain both GTA structural cluster and regulatory gene homologues. The evolutionary relationships of GTA structural proteins to (pro)phage proteins indicated by the phylogenetic tree patterns suggest a predominantly vertical descent of GTA-like sequences in the a-proteobacteria and little past gene exchange with (pro)phages. [ABSTRACT FROM AUTHOR]

Introduction and aim: Brucellosis is a zoonotic disease. Experimental clinical and laboratory diagnosis is still facing problems in identifying the organism. The present study will diagnose a Brucella infection in camel blood in Qatar using serological assays. Isolation and identification were performed on a camel blood sample. Brucella in bacterial isolates was determined by real-time polymerase chain reaction (RT-PCR) as a gold standard test. Material and methods: A total of 220 samples, 200 random serum samples, and 20 EDTA blood samples were selected among the above-mentioned random samples, and 20 serum samples from camel handlers were collected from Al Shahaniya province, Qatar. The Rose Bengal test (RBT), buffered antigen plate agglutination test (BAPAT), and enzyme linked immunosorbent assay (cELISA) for the monoclonal antibody in serum samples were performed using commercially available kits. For the molecular detection of Brucella, conventional PCR and real-time PCR (GPS kit) were used for the genus-specific insertion sequence IS711. Brucella melitensis (MICROBOSS Hightech GmbH kit) was used to identify subspecies. Results: The results identified by vitek2 compact (30%) showed B. melitensis in 6 samples out of 20 isolates. Both conventional (66.67%) and RT-PCR (83.33%) analyses supported this, demonstrating the presence of Brucella. These tests also showed that Brucella species were present in Rose Bengal 182/200 (91%), BAPAT 182/200 (91%), and cELISA (90%) 180/200 in camel serum. Concusion: To conclude, the prevalence of brucellosis in dromedary camels is higher in this region, and as a matter of urgency, measures should be taken to control the disease. [ABSTRACT FROM AUTHOR]

Background: Brucella melitensis is recognized as one of the predominant zoonotic pathogens globally. Live-attenuated vaccine Rev 1 is currently the most effective vaccine for controlling B. melitensis in small ruminants. While Brucella inactivated, nanoparticle, and subunit vaccines are less effective and require multiple doses, live-attenuated vaccines are less expensive and more efficacious. Several drawbacks are associated with the administration of current attenuated B. melitensis vaccines, including interference with serological diagnostic tests, inducing abortion in pregnant animals, shedding in milk, and zoonotic infections in humans. In this systematic review, we summarize the current literature (1970–2022) on B. melitensis vaccines and review their advantages and disadvantages in order to support the rationale for a need for new or improved small ruminant brucellosis vaccines. Methods: A systematic search was carried out in Web of Science, CAB Abstracts, and PubMed. The original articles describing the B. melitensis vaccines were included. Review articles, articles not published in English, articles that did not offer full text, editorials, correspondences, case reports, case series, diagnostic tests, duplicate publications, and other Brucella vaccines (e.g., B. abortus and B. suis) were excluded. Results: Out of 3700 studies, we identified 18 articles that evaluated B. meltensis vaccines, including recombinant B. melitensis strains (16MΔhfq, 16MΔTcfSR, M5-90ΔmanB, LVM31, M5-90ΔvjbR, 16MΔmucR, ΔznuA, M5-90Δpgm, M5-90ΔwboA), live B. melitensis strain (Rev 1), nanoparticle vaccines (B. melitensis 16M, B. melitensis OMP 31, FliC protein—Mannosylated Chitosan Nanoparticles (FliC and FliC-MCN), B. melitensis and B. abortus combined, and B. melitensis 16M nanoparticles combined with oligopolysaccharide), subunit vaccines (outer membrane vesicles or outer membrane proteins), and a DNA vaccine based on B. melitensis outer membrane proteins (Omp25 and Omp31). The results from these studies revealed that these vaccines can induce humoral and cellular responses and reduce macrophage survival. However, most of these vaccines were evaluated only in murine models, which may not accurately reflect how they work in natural hosts. Conclusions: The high prevalence of B. melitensis in humans and animals remains an issue in many parts of the world. Human brucellosis can be prevented by controlling brucellosis in livestock using vaccination and test-and-removal strategies. Prospective vaccines have limitations, including interference with serodiagnostics after vaccination, virulence in humans and animals, the requirement of booster vaccinations, and insufficient efficacy in preventing infection or abortion. Moreover, most of these vaccines have been assessed in mice models, which have failed to predict immunogenicity or efficacy in natural hosts. Because of these limitations and the re-emergence of B. melitensis worldwide with a high incidence of human infection, our review suggests a need for additional research into the molecular pathology and immunological properties of B. melitensis infection and the identification of protective epitopes or genes that would allow for the development of improved vaccines for small ruminants. [ABSTRACT FROM AUTHOR]

Westphal's procedure was found the most suitable for the isolation of lipopolysaccharide fractions from Brucella melitensis cells; only the fraction obtained in the aqueous layer (LPS) is studied in this paper. Beside LPS, an extracellular polysaccharide fraction (PS) was extracted from the culture medium of the bacteria, and the chemical composition of both fractions, LPS and PS, has been compared. In both cases, nucleic acid contaminants were eliminated either by alcohol precipitation or by use of cetavlon. These treatments also removed an acidic polysaccharide containing mainly galactosaminuronic acid, which might be Vi antigen. The main difference between LPS and PS is the presence of a small amount of lipid A in LPS (ca. 1%). As in the case of lipid A isolated from LPS of Enterobacteria, Brucella lipid A contains glucosamine and fatty acids, but β-hydroxymyristic acid could not be detected. The behaviour of LPS and PS fractions on DEAE-cellulose columns is very similar, and the small differences that could be observed may be explained by solubility changes due to the lipid moiety present in LPS. Both fractions contain 2-keto-3-hydroxy-octulosonic acid, a mixture of D- and L-glyceroD-mannoheptoses, a high amount of glucose, galactose, a 3,6-dideoxyhexose (colitose or abequose), and a methylated sugar behaving as a methylated derivative of dideoxyhexose. Small amounts of rhamnose in PS and of arabinose in LPS seem to be due to contaminants. [ABSTRACT FROM AUTHOR]

Background: Brucellosis is a serious public health problem distributed worldwide. Jordan has one of the highest incidences of brucellosis globally, mostly caused by infection with Brucella melitensis, which is associated with small, ruminant animals such as sheep. Sheep are an important livestock species in Jordan where there is a high value is placed on keeping animals at home. The incidence of human brucellosis rapidly increased following the Syrian conflict, in association with the resettlement of displaced people. Methods: A systems thinking (ST) approach was applied to develop a model to explain the dynamics of brucellosis in Jordan. A causal loop diagram (CLD) was developed to visualize and conceptualize interactions and feedback between several factors involved in sheep husbandry, animal and human health, and livestock trading systems. The CLD was constructed using information from published literature, historical governmental reports, policy documents and media coverage regarding brucellosis in Jordan. Semi-structured, in-depth interviews with diverse stakeholders were conducted to elicit information on their understanding of critical factors associated with brucellosis. Results: The CLD demonstrated that brucellosis transmission in Jordan is driven by sheep husbandry practices like livestock movement/trading and the management of symptomatic animals (those suffering abortion). Five dimensions were identified to be associated with brucellosis transmission: human infection; consumers; traders and markets; and livestock infection and control. Conclusion: The development of a CLD to explain brucellosis transmission in Jordan provides a better understanding of and reveals the multi-sectoral nature of the problem. The need for a multi-sectoral approach for effective brucellosis management in Jordan is clear from the five dimensions identified. [ABSTRACT FROM AUTHOR]

Brucellosis is a disease caused by the bacterium Brucella and typically transmitted through contact with infected ruminants. It is one of the most common chronic zoonotic diseases and of particular interest to public health agencies. Despite its well-known transmission history and characteristic symptoms, we lack a more complete understanding of the evolutionary history of its best-known species—Brucella melitensis. To address this knowledge gap we fortuitously found, sequenced and assembled a high-quality ancient B. melitensis draft genome from the kidney stone of a 14th-century Italian friar. The ancient strain contained fewer core genes than modern B. melitensis isolates, carried a complete complement of virulence genes, and did not contain any indication of significant antimicrobial resistances. The ancient B. melitensis genome fell as a basal sister lineage to a subgroup of B. melitensis strains within the Western Mediterranean phylogenetic group, with a short branch length indicative of its earlier sampling time, along with a similar gene content. By calibrating the molecular clock we suggest that the speciation event between B. melitensis and B. abortus is contemporaneous with the estimated time frame for the domestication of both sheep and goats. These results confirm the existence of the Western Mediterranean clade as a separate group in the 14th CE and suggest that its divergence was due to human and ruminant co-migration. Author summary: We describe the reconstruction of a high-quality ancient B. melitensis genome isolated from the kidney stone of a 14th C. CE Italian friar. We confirm the presence of a B. melitensis genome from the Western Mediterranean clade in Italy during this time period and refine previous estimates for the diversification of this phylogenetic group. In addition, we propose that the speciation event separating B. abortus and B. melitensis occurred during the domestication of sheep and goats approximately 9000 BCE. By comparing the gene content of the ancient B. melitensis genome with modern isolates, we provide evidence that neither the B. melitensis phylogenetic groups nor their respective sequence types necessarily correspond with the genetic components of their strains. The accessory genome of B. melitensis appears to be influenced by its geographic location, more so than its sequence type. We propose splitting the Eastern Mediterranean clade into three smaller groups to better reflect these genomic differences. [ABSTRACT FROM AUTHOR]

Iron is an essential element required for all organisms. Iron response regulator (Irr) is a crucial transcriptional regulator and can affect the growth and iron uptake of Brucella. The growth rate of Brucella melitensis M5-90 irr mutant was significantly lower than that of B. melitensis M5-90 under normal or iron-sufficient conditions, however, the growth rate of the B. melitensis M5-90 irr mutant was significantly higher than that of B. melitensis M5-90 under iron-limited conditions. In addition, irr mutation significantly reduced iron uptake under iron-limited conditions. Previous studies suggested that the Irr protein has multiple target genes in the Brucella genome that are involved in iron metabolism. Therefore, in the present study, a Dap-seq approach was used to investigate the other iron metabolism genes that are also regulated by the Irr protein in Brucella. A total of seven genes were identified as target genes for Irr in this study and the expression levels of these seven genes were similar in both the RNA-seq and qRT-PCR results. The electrophoretic mobility shift assay confirmed that six out of the seven genes, namely rirA (BME_RS13665), membrane protein (BME_RS01725), hypothetical protein (BME_RS09560), ftrA (BME_RS14525), cation-transporting P-type ATPase (zntA) (BME_RS10660), and 2Fe-2S binding protein (BME_RS13655), interact with the Irr protein. Furthermore, the iron utilization and growth assay experiments confirmed that rirA was involve in iron metabolism and growth of Brucella. In summary, our results identified six genes regulated by the Irr protein that may participate in iron metabolism, and the rirA was identified as a regulon of Irr and it also plays a role in iron metabolism of Brucella. Collectively, these results provide valuable insights for the exploration of Brucella iron metabolism. Author summary: Iron response regulator (Irr) plays a key role in iron metabolism of Brucella. In this study, the Brucella melitensis M5-90 irr mutant appeared significant different of growth and iron utilization rate compared with that of the Brucella melitensis M5-90 strain. However, the specific binding sites of Irr in Brucella are not fully understood. Here, we used Dap-seq to identify the potential target genes of Irr in the genome of Brucella melitensis M5-90. A total of seven genes were identified as target genes for Irr and the expression of levels of these seven genes were identified using qRT-PCR. In addition, Six out of seven genes namely rirA, membrane protein, hypothetical protein, ftrA, zntA and 2Fe-2S binding protein interact with the Irr protein. Furthermore, the mutation of rirA markedly affected the iron utilization and growth rate of Brucella. Overall, these results provide valuable insights for the exploration of Brucella iron metabolism. [ABSTRACT FROM AUTHOR]

Background: Brucellosis is one of the most important zoonotic diseases caused by Gram-negative bacteria belonging to the genus Brucella. Detection of Brucella species in different countries is of utmost importance. Aims: This study aimed to detect Brucella abortus and Brucella melitensis in domestic ruminant blood samples and their ticks in western Iran. Methods: Sampling was conducted on ruminants from August to September 2020 in four different counties of Kurdistan Province, including Divandareh, Marivan, Baneh, and Sanandaj. Totally, 250 blood samples were collected from 250 small ruminants. There were no ticks on the skin of six (2.4%) ruminants, and 244 ticks were isolated from 244 animals. After genomic DNA extraction from all the collected samples, quantitative polymerase chain reaction (qPCR) was performed to detect IS711 gene. Results: Based on qPCR results, Brucella genus was detected in two blood samples (0.8%) from female sheep and four ticks (1.6%) from male sheep, including three Dermacentor marginatus (1.22%) and one Rhipicephalus turanicus (0.4%). Although B. melitensis was not detected in any tick or blood sample, one tick sample (D. marginatus) was positive for B. abortus. Conclusion: Considering the positivity of ticks for brucellosis in this study, there is a possibility of Brucella transmission from infected ticks to humans and animals through tick bites, nevertheless, in order to identify the Brucella transmission relationship between ticks and animals, serological tests should be used in future studies. [ABSTRACT FROM AUTHOR]