Expression of cytokine and Apoptosis-Associated genes in mice bone Marrow-Derived Macrophages stimulated with Brucella recombinant type IV secretion effectors.

BACKGROUND: Brucellosis is an economically important infectious caused by most commonly by Brucella. Detection of infected animals at the early stage is important for controlling the disease. The diagnostic antigens, usually protein antigens, have attracted much interest. However, the accurate mechanism of immune response is still unknown. The secretory effectors (BPE005, BPE275, and BPE123) of the type IV secretion system (T4SS) were involved in the intracellular circulation process of Brucella and the immune responses of the host. METHODS: Genes encoding three B. abortus effector proteins (BPE005, BPE275, and BPE123) of T4SS were cloned and the recombinant proteins were expressed and purified. The purified recombinant proteins were named rBPE005, rBPE275 and rBPE123. Then, the expressions of Th1- and Th2-related cytokine genes were analyzed in mice bone marrow-derived macrophages (BMDMs) after stimulation with rBPE005, rBPE275, and rBPE123. Furthermore, four apoptosis-associated genes (Caspase-3, Caspase-8, Bax, and Bcl-2) were also detected to explore the damage of the proteins to the cells. RESULTS: Expressions of all Th1- and Th2-related cytokine genes were induced with three proteins, and different cytokine expression patterns induced by each protein depend on the stimulation time and dose of protein. However, expressions of apoptosis-related genes did not change. CONCLUSION: These results showed that the secreted antigens of Brucella induced an immune reaction via the production of Th1- and Th2-type cytokines in BMDMs without exerting any damage on the cells.

Detection and quantification of Brucella abortus DNA in water buffaloes (bubalus bubalis) using droplet digital polymerase chain reaction.

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of Brucella spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of Brucella abortus in buffalo tissue samples. The protocol was validated using proficiency test samples for Brucella spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect B. abortus in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.

Development of a novel multi-epitope vaccine for brucellosis prevention.

Brucellosis, a zoonotic disease caused by Brucella, presents a significant threat to both animal and human health. In animals, the disease can lead to infertility, miscarriage, and high fever, while in humans, symptoms may include recurrent fever, fatigue, sweating, hepatosplenomegaly, and joint and muscle pain following infection. Treatment often involves long-term antibiotic therapy, placing a substantial psychological and financial burden on patients. While vaccination is crucial for prevention, current animal vaccines have drawbacks such as residual virulence, and a safe and effective human vaccine is lacking. Hence, the development of a vaccine for brucellosis is imperative. In this study, we utilized bioinformatics methods to design a multi-epitope vaccine targeting Brucella. Targeting Heme transporter BhuA and polysaccharide export protein, we identified antigenic epitopes, including six cytotoxic T lymphocyte (CTL) dominant epitopes, six helper T lymphocyte (HTL) dominant epitopes, one conformation B cell dominant epitope, and three linear B cell dominant epitopes. By linking these epitopes with appropriate linkers and incorporating a Toll-like receptor (TLR) agonist (human beta-defensin-2) and an auxiliary peptide (Pan HLA-DR epitopes), we constructed the multi-epitope vaccine (MEV). The MEV demonstrated high antigenicity, non-toxicity, non-allergenicity, non-human homology, stability, and solubility. Molecular docking analysis and molecular dynamics simulations confirmed the interaction and stability of the MEV with receptors (MHCI, MHCII, TLR4). Codon optimization and in silico cloning validated the translation efficiency and successful expression of MEV in Escherichia coli. Immunological simulations further demonstrated the efficacy of MEV in inducing robust immune responses. In conclusion, our findings suggest that the engineered MEVs have the potential to stimulate both humoral and cellular immune responses, offering valuable insights for the future development of safe and efficient Brucella vaccines.

Analysis of the Brucella melitensis epidemic in Xinjiang: genotyping, polymorphism, antibiotic resistance and tracing.

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.

Hemophagocytic Lymphohistiocytosis Induced by Brucellosis: A Case Report.

Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory condition triggered by infections, malignancies, or autoimmune conditions. Brucellosis is a zoonotic disease contracted through exposure to infected animals or consumption of unpasteurized dairy products. The complications of both pathologies may be fatal. This report presents a rare instance of HLH induced by Brucellosis, highlighting the need for increased recognition of this life-threatening association.

Characterization of the adaptive cellular and humoral immune responses to persistent colonization of Brucella abortus strain RB51 in a Jersey cow.

Brucella abortus strain RB51 is the commercial cattle vaccine used in the United States (US) and many parts of the world against bovine brucellosis. RB51 was licensed for use in 1996, and it has been shown to be safe and efficacious in cattle, eliciting humoral and cellular responses in calves and adult animals. In 2017, an epidemiological trace-back investigation performed by the Centers for Disease Control and Prevention (CDC) identified human cases of brucellosis caused by infection with RB51. These infections resulted from the consumption of unpasteurized dairy products, which were traced back to otherwise healthy animals that were shedding RB51 in their milk. At the current time, six adult Jersey cows have been identified in the U.S. that are shedding RB51 in milk. One of the RB51 shedding cattle was obtained and housed at the National Animal Disease Center (NADC) for further study. Improved understanding of host cellular and humoral immune responses to RB51 in persistently colonized cattle may be achieved by the characterization of responses in shedding animals. We hypothesized, based on the lack of RB51 clearance, that the RB51 shedder animal has a diminished adaptive cellular immune response to RB51. Our data demonstrate that in the presence of persistent RB51 infection, there is a lack of peripheral anti-RB51 CD4+ T cell responses and a concurrently high anti-RB51 IgG humoral response. By understanding the mechanisms that result in RB51 persistence, the development of improved interventions or vaccinations for brucellosis may be facilitated, which would provide public health benefits, including reducing the risks associated with the consumption of non-pasteurized milk products.

Zoonotic Bacterial Infections Triggering Cytokine Storm Syndrome.

Zoonotic infections can result in life-threatening complications that can manifest with hemophagocytic lymphohistiocytosis (HLH)/cytokine storm syndrome (CSS). Bacteria constitute the largest group of zoonotic infection-related HLH cases. The growing list of zoonotic bacterial infections associated with HLH/CSS include Brucella spp., Rickettsia spp., Ehrlichia, Coxiella burnetii, Mycobacterium spp., and Bartonella spp. Patients most commonly present with fever, cytopenias, hepatosplenomegaly, myalgias, and less frequently with rash, jaundice, and lymphadenopathy.

Single-cell landscape revealed immune characteristics associated with disease phases in brucellosis patients.

A comprehensive immune landscape for Brucella infection is crucial for developing new treatments for brucellosis. Here, we utilized single-cell RNA sequencing (scRNA-seq) of 290,369 cells from 35 individuals, including 29 brucellosis patients from acute (n = 10), sub-acute (n = 9), and chronic (n = 10) phases as well as six healthy donors. Enzyme-linked immunosorbent assays were applied for validation within this cohort. Brucella infection caused a significant change in the composition of peripheral immune cells and inflammation was a key feature of brucellosis. Acute patients are characterized by potential cytokine storms resulting from systemic upregulation of S100A8/A9, primarily due to classical monocytes. Cytokine storm may be mediated by activating S100A8/A9-TLR4-MyD88 signaling pathway. Moreover, monocytic myeloid-derived suppressor cells were the probable contributors to immune paralysis in acute patients. Chronic patients are characterized by a dysregulated Th1 response, marked by reduced expression of IFN-γ and Th1 signatures as well as a high exhausted state. Additionally, Brucella infection can suppress apoptosis in myeloid cells (e.g., mDCs, classical monocytes), inhibit antigen presentation in professional antigen-presenting cells (APCs; e.g., mDC) and nonprofessional APCs (e.g., monocytes), and induce exhaustion in CD8+ T/NK cells, potentially resulting in the establishment of chronic infection. Overall, our study systemically deciphered the coordinated immune responses of Brucella at different phases of the infection, which facilitated a full understanding of the immunopathogenesis of brucellosis and may aid the development of new effective therapeutic strategies, especially for those with chronic infection.

Meta-genomic next-generation sequencing in the diagnosis of brucellosis: Five cases from a non-endemic area.

Metagenomic next-generation sequencing (mNGS) in diagnosis of human brucellosis is comparatively unexplored. This report details five human brucellosis cases diagnosed using mNGS based on Illumina sequencing platform, comprising three females and two males, four with epidemiological exposure. In cases 1 and 2, plasma mNGS results showed one positive and one negative for Brucella melitensis, and subsequent blood cultures were both positive. Cases 3, 4 and 5 involved spinal brucellosis, some with paravertebral abscesses. mNGS from infectious tissue samples successfully detected Brucella, with read counts ranging between 30 and 1314, yet cultures were negative in cases 4 and 5. Following antibiotic and surgical treatments, all patients showed clinical improvement. This report shows mNGS testing enhances the detection sensitivity of brucellosis diagnosis.

What is this summary about? Brucella is a type of bacteria that can infect humans and animals. It causes a disease called brucellosis. Symptoms of brucellosis include fever and fatigue, among others. Meta-genomic next-generation sequencing (mNGS) is a tool for sequencing the DNA of bacteria. In this report, we use mNGS to diagnose human brucellosis in five cases.What were the results? Brucella was found in the blood of two infected people, but mNGS found Brucella in only one. Of three people with Brucella infection of the spine, mNGS found Brucella in the infected tissue but Brucella was only cultured in one case. Following antibiotic and surgical treatments, all five patients showed improvement of their symptoms.What do the results of the study mean? mNGS is a relatively rapid and effective diagnostic method that can improve the detection of Brucella in brucellosis.

Multiple pathogens co-exposure and associated risk factors among cattle reared in a wildlife-livestock interface area in Kenya.

Introduction: Understanding multi-pathogen infections/exposures in livestock is critical to inform prevention and control measures against infectious diseases. We investigated the co-exposure of foot-and-mouth disease virus (FMDV), Brucella spp., Leptospira spp., and Coxiella burnetii in cattle in three zones stratified by land use change and with different wildlife-livestock interactions in Narok county, Kenya. We also assessed potential risk factors associated with the transmission of these pathogens in cattle. Methods: We identified five villages purposively, two each for areas with intensive (zone 1) and moderate wildlife-livestock interactions (zone 2) and one for locations with low wildlife-livestock interactions (zone 3). We sampled 1,170 cattle from 390 herds through a cross-sectional study and tested the serum samples for antibodies against the focal pathogens using enzyme-linked immunosorbent assay (ELISA) kits. A questionnaire was administered to gather epidemiological data on the putative risk factors associated with cattle's exposure to the investigated pathogens. Data were analyzed using the Bayesian hierarchical models with herd number as a random effect to adjust for the within-herd clustering of the various co-exposures among cattle. Results: Overall, 88.0% (95% CI: 85.0-90.5) of the cattle tested positive for at least one of the targeted pathogens, while 41.7% (95% CI: 37.7-45.8) were seropositive to at least two pathogens. FMDV and Brucella spp. had the highest co-exposure at 33.7% (95% CI: 30.9-36.5), followed by FMDV and Leptospira spp. (21.8%, 95% CI: 19.5-24.4), Leptospira spp. and Brucella spp. (8.8%, 95% CI: 7.2-10.6), FMDV and C. burnetii (1.5%, 95% CI: 0.7-2.8), Brucella spp. and C. burnetii (1.0%, 95% CI: 0.3-2.2), and lowest for Leptospira spp. and C. burnetii (0.3%, 95% CI: 0.0-1.2). Cattle with FMDV and Brucella spp., and Brucella spp. and Leptospira spp. co-exposures and those simultaneously exposed to FMDV, Brucella spp. and Leptospira spp. were significantly higher in zone 1 than in zones 2 and 3. However, FMDV and Leptospira spp. co-exposure was higher in zones 1 and 2 than zone 3. Discussion/conclusion: We recommend the establishment of a One Health surveillance system in the study area to reduce the morbidity of the targeted zoonotic pathogens in cattle and the risks of transmission to humans.

New synthesis of oligosaccharides modelling the M epitope of the Brucella O-polysaccharide.

Brucellosis is a dangerous zoonotic disease caused by bacteria of the genus Brucella. Diagnosis of brucellosis is based on the detection in animal and human sera of antibodies to the O-polysaccharide of Brucella lipopolysaccharide. The currently employed serodiagnosis of brucellosis relies on the use of the Brucella O-polysaccharide as a diagnostic antigen. However, the existence of bacterial species, which also express O-polysaccharides structurally similar to that of Brucella, may decrease the specificity of the brucellosis detection due to false-positive test results. It has been shown that the efficiency of the test can be significantly improved by using synthetic oligosaccharides that correspond to the so-called M epitope of the Brucella O-antigen. This epitope is characterized by an α-(1→3)-linkage between d-perosamine units and is unique to Brucella. Here we report on an efficient approach to the synthesis of oligosaccharides that model the M epitope of the Brucella O-polysaccharide. The approach is based on the use of the α-(1→3)-linked disaccharide thioglycoside as the key donor block. Its application allowed the straightforward assembly of a set of four protected oligosaccharides, which includes a disaccharide, two trisaccharides, and a tetrasaccharide, in five glycosylation steps. The synthesized oligosaccharides are planned to be used in the development of diagnostic tools for identifying brucellosis in humans and domestic animals, as well as a potential vaccine against it.

Molecular epidemiology of Brucella abortus isolated from the environment in Ningxia Hui autonomous region, China.

Brucellosis is among the key zoonotic infectious diseases in China, and The Ningxia Hui Autonomous Region represents a major endemic area, and it is one of the main causes of poverty in the region due to illness. In Ningxia, there is substantial research on Brucella melitensis, studies on the molecular epidemiology of Brucella abortus are notably scarce. Consequently, this study aims to undertake pathogenic isolation and molecular epidemiological research on Brucella abortus isolated from the environment in Ningxia, providing insights and evidence to advance the prevention and control measures for brucellosis in the region. Building on traditional pathogenic detection methods, this research employs whole-genome sequencing(WGS) techniques and bioinformatics software to conduct a phylogenetic comparison of Ningxia strains and strains of Brucella abortus from various geographical origins. The results indicate that four Brucella abortus strains are classified as biovar 3 and MLST type ST2. It is shown that the local strains were closer phylogenetic relationships with strains from Asian and European countries. The presence of Brucella abortus in certain environmental sectors of Ningxia indicates a risk of transmission from the environment to animals and subsequently to humans. In conclusion, the Brucella abortus exists in some farming environments in Ningxia, and exists for a long time. Therefore, it is necessary to strengthen the monitoring of the disinfection effect of the farming environment to provide a basis for the forward movement of the gate of brucellosis prevention and control.

Draft genome sequences of Brucella melitensis from human and animal sources in India.

We present the draft genome sequences of 23 Brucella melitensis isolates derived from human and animal sources across India with genome size predominantly at 3.207 M and uniform GC content (57.24%) across isolates. The accession numbers and detailed sequencing data enhance the utility of this resource for further genomic studies.

Investigation of the Seroprevalence of Brucella Antibodies and Characterization of Field Strains in Immunized Dairy Cows by B. abortus A19.

(1) Background: One method of eradicating brucellosis is to cull cattle that test positive for antibodies 12 months after being vaccinated with the 19-strain vaccine. Variations in immunization regimens and feeding practices may contribute to differences in the rate of persistent antibodies. We conducted this study to investigate the real positive rate of Brucella antibody in field strains of Brucella spp. after immunization over 12 months in dairy cows. This research aims to provide data to support the development of strategies for preventing, controlling, and eradicating brucellosis. (2) Method: We employed the baseline sampling method to collect samples from cows immunized with the A19 vaccine for over 12 months in Lingwu City from 2021 to 2023. Serological detection was conducted using the RBPT method. An established PCR method that could distinguish between 19 and non-19 strains of Brucella was utilized to investigate the field strains of Brucella on 10 dairy farms based on six samples mixed into one using the Mathematical Expectation strategy. (3) Results: We analyzed the rates of individual seropositivity and herd seropositive rates in dairy cattle in Lingwu City from 2021 to 2023 and revealed that antibodies induced by the Brucella abortus strain A19 vaccine persist in dairy herds for more than 12 months. We established a PCR method for identifying both Brucella A19 and non-A19 strains, resulting in the detection of 10 field strains of Brucella abortus from 1537 dairy cows. By employing a Mathematical Expectation strategy, we completed testing of 1537 samples after conducting only 306 tests, thereby reducing the workload by 80.1%. (4) Conclusions: There was a certain proportion of cows with a persistent antibody titer, but there was no evidence that all of these cattle were naturally infected with Brucella. The established PCR method for distinguishing between Brucella abortus strain 19 and non-19 strains can be specifically utilized for detecting natural Brucella infection in immunized cattle. We propose that relying solely on the detection of antibodies in cattle immunized with the A19 vaccine more than 12 months previously should not be solely relied upon as a diagnostic basis for brucellosis, and it is essential to complement this approach with PCR analysis to specifically identify field Brucella spp. Brucella abortus was the predominant strain identified in the field during this study. Detection based on the Mathematical Expectation strategy can significantly enhance detection efficiency.

Transmission of Brucella canis in a canine kennel following introduction of an infected dog.

Brucella canis is a zoonotic pathogen and the main causative agent of canine brucellosis. In the Netherlands, B. canis had previously only been detected in individual cases of imported dogs. However, an outbreak of B. canis occurred for the first time in a cohort of autochthonous dogs in a breeding kennel in 2019. The outbreak began with a positive serological test result of an imported intact male dog showing clinical symptoms of brucellosis. Consequently, urine and blood samples were collected and tested positive for B. canis by culture, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) and whole-genome-sequencing (WGS). Screening of the contact dogs in the kennel where the index case was kept, revealed that antibodies against B. canis could be detected in 23 out of 69 dogs (34 %) by serum agglutination test (SAT). Of the 23 seropositive dogs, B. canis could be cultured from the urine and/or heparin samples of 19 dogs (83 %). This outbreak represents the first documented case of transmission of B. canis to autochthonous contact dogs in the Netherlands. WGS revealed all B. canis isolates belonged to the same cluster, which means the transmission of B. canis in the breeding kennel was most likely caused by the introduction of one infected dog. Comparing this cluster with data from other B. canis isolates, it also appears that characteristic clusters of B. canis are present in several endemic countries. These clusters seem to remain stable over time and may help in locating the origin of new isolates found. This outbreak showed that the international movement of dogs from endemic countries poses a threat to the canine population, while serological screening and WGS proved to be valuable tools for respectively screening and the epidemiological investigation.

Establishment of an A/T-Rich Specifically MGB Probe digital droplet PCR Assays Based on SNP for Brucella wild strains and vaccine strains.

In recent years, immunization with the S2 live-attenuated vaccine has been recognized as the most economical and effective strategy for preventing brucellosis in Inner Mongolia, China. However, there are still challenges related to vaccine toxicity and the inability to distinguish between vaccine immunization and natural infection. Therefore, in this study, we developed a digital droplet polymerase chain reaction (ddPCR) assay based on single-nucleotide polymorphism (SNP) loci to identify wild Brucella strains and S2 vaccine strains. The assay demonstrated excellent linearity (R2> 0.99) with a lower detection limit of 10 copies/µL for both wild and vaccine strains. Additionally, the ddPCR assay outperformed the real-time fluorescent quantitative PCR (qPCR) assay in screening 50 clinical samples. We have established an effective and highly sensitive ddPCR assay for Brucella, providing an efficient method for detecting and differentiating wild strains of Brucella from the S2 vaccine strain.

Development and Bayesian validation of a competitive inhibition ELISA for detection of antibodies against Brucella abortus in cattle.

Bovine brucellosis is a zoonotic disease caused by Brucella abortus, responsible for abortions in cows. It is endemic in low- and middle-income countries, where the brucellosis control and eradication programs are based on compulsory vaccination, detection of infected cattle through serologic assays, and culling of infected animals at slaughterhouses. The development of high sensitivity and specificity, and low-cost serologic assays guarantee their implementation in countries where the disease is endemic. The aim of the present study was to develop and validate a competitive inhibition enzyme-linked immune assay (ciELISA) to detect anti-B. abortus antibodies in sera from cattle. The developed ciELISA was validated using 2833 serum samples from dairy and beef cattle. From these, 1515 sera were from uninfected cows that belonged to free of brucellosis herds and 1318 were from infected cows that belonged positive to brucellosis herds. Sera were analyzed with the developed ciELISA, the buffer plate antigen (BPA) test, and the complement fixation test (CFT). The brucellosis status of the herds was officially established according to the country legislation and consistent for at least 5 years and was defined for each cow using the CFT as gold standard. The cutoff for the ciELISA was calculated using a ROC curve and its sensitivity and specificity were analyzed using the Bayesian Latent Class Model (BLCM) approach. The agreement among tests was calculated using the kappa (κ) value. In addition, 15 calves were vaccinated with 3 × 1010 viable cells of B. abortus Strain 19 vaccine, and the dynamics of antibodies were measured by CFT, buffered plate antigen (BPA) test, and the developed ciELISA. The obtained cutoff for ciELISA was ≥ 47 percentage of inhibition (% I), at the BLCM approach the sensitivity was 99.01 % (95 % CI: 97.55-100) and the specificity 98.74 % (95 % CI: 97.68-99.8). The κ between the ciELISA and BPA was κ = 0.88 and between the ciELISA and CFT κ = 0.95. Antibodies against B. abortus were detected in all the vaccinated calves 7 days after vaccination (AV) by the three assays, at day 135 AV all the calves were negative to CFT (15/15), 93.3 % (14/15) to ciELISA and 73.3 % (11/15) to BPA, and at day 190 AV all the calves were negative to the three assays. The developed ciELISA showed a very good performance, could detect the majority of vaccinated animals as negative after 135 days and could be used for the detection of anti-B. abortus antibodies in serum samples for the brucellosis control and eradication program.

Purification and characterization of the produced hyaluronidase by Brucella Intermedia MEFS for antioxidant and anticancer applications.

Hyaluronidase (hyase) is an endoglycosidase enzyme that degrades hyaluronic acid (HA) and is mostly known to be found in the extracellular matrix of connective tissues. In the current study, eleven bacteria isolates and one actinomycete were isolated from a roaster comb and screened for hyase production. Seven isolates were positive for hyase, and the most potent isolate was selected based on the diameter of the transparent zone. Based on the morphological, physiological, and 16 S rRNA characteristics, the most potent isolate was identified as Brucella intermedia MEFS with accession number OR794010. The environmental conditions supporting the maximum production of hyase were optimized to be incubation at 30 ºC for 48 h and pH 7, which caused a 1.17-fold increase in hyase production with an activity of 84 U/mL. Hyase was purified using a standard protocol, including precipitation with ammonium sulphate, DEAE as ion exchange chromatography, and size exclusion chromatography using Sephacryle S100, with a specific activity of 9.3-fold compared with the crude enzyme. The results revealed that the molecular weight of hyase was 65 KDa, and the optimum conditions for hyase activity were at pH 7.0 and 37 °C for 30 min. The purified hyase showed potent anticancer activities against colon, lung, skin, and breast cancer cell lines with low toxicity against normal somatic cells. The cell viability of hyase-treated cancer cells was found to be in a dose dependent manner. Hyase also controlled the growth factor-induced cell cycle progression of breast cancer cells and caused relative changes in angiogenesis-related genes as well as suppressed many pro-inflammatory proteins in MDA cells compared with 5-fluorouracil, indicating the significant role of hyase as an anticancer agent. In addition, hyase recorded the highest DPPH scavenging activity of 65.49% and total antioxidant activity of 71.84% at a concentration of 200 µg/mL.

A scoping review on the epidemiology and public significance of Brucella abortus in Chinese dairy cattle and humans.

Brucellosis, caused by Brucella spp., is a re-emerging One Health disease with increased prevalence and incidence in Chinese dairy cattle and humans, severely affecting animal productivity and public health. In dairy cattle, B. abortus is the primary causative agent although infections with other Brucella species occur occasionally. However, the epidemiological and comparative importance of B. abortus in dairy cattle and humans remains inadequately understood throughout China due to the heterogeneity in locations, quality, and study methods. This scoping review aims to describe the changing status of B. abortus infection in dairy cattle and humans, investigate the circulating Brucella species and biovars, and identify factors driving the disease transmission by retrieving publicly accessible literature from four databases. After passing the prespecified inclusion criteria, 60 original articles were included in the final synthesis. Although the reported animal-level and farm-level prevalence of brucellosis in dairy cattle was lower compared to other endemic countries (e.g. Iran and India), it has been reported to increase over the last decade. The incidence of brucellosis in humans displayed seasonal increases. The Rose Bengal Test and Serum Agglutination Test, interpreted in series, were the most used serological test to diagnose Brucella spp. in dairy cattle and humans. B. abortus biovar 3 was the predominant species (81.9%) and biovar (70.3%) in dairy cattle, and B. melitensis biovar 3 was identified as the most commonly detected strain in human brucellosis cases. These strains were mainly clustered in Inner Mongolia and Shannxi Province (75.7%), limiting the generalizability of the results to other provinces. Live cattle movement or trade was identified as the key factor driving brucellosis transmission, but its transmission pattern remains unknown within the Chinese dairy sector. These knowledge gaps require a more effective One Health approach to be bridged. A coordinated and evidence-based research program is essential to inform regional or national control strategies that are both feasible and economical in the Chinese context.

Goat milk as the connecting link in dengue and brucella coinfection.

In areas with widespread prevalence of myth of goat milk as a platelet booster, the goat milk can be connecting link in dengue and brucella coinfection.