Deletion of the alr gene in Brucella suis S2 attenuates virulence by enhancing TLR4-NF-κB-NLRP3- mediated host inflammatory responses.

Brucella is an intracellular parasitic bacterium lacking typical virulence factors, and its pathogenicity primarily relies on replication within host cells. In this study, we observed a significant increase in spleen weight in mice immunized with a Brucella strain deleted of the gene for alanine racemase (Alr), the enzyme responsible for alanine racemization (Δalr). However, the bacterial load in the spleen markedly decreased in the mutant strain. Concurrently, the ratio of white pulp to red pulp in the spleen was increased, serum IgG levels were elevated, but no significant damage to other organs was observed. In addition, the inflammatory response was potentiated and the NF-κB-NLRP3 signaling pathway was activated in macrophages (RAW264.7 Cells and Bone Marrow-Derived Cells) infect ed with the Δalr mutant. Further investigation revealed that the Δalr mutant released substantial amounts of protein in a simulated intracellular environment which resulted in heightened inflammation and activation of the TLR4-NF-κB-NLRP3 pathway in macrophages. The consequent cytoplasmic exocytosis reduced intracellular Brucella survival. In summary, cytoplasmic exocytosis products resulting from infection with a Brucella strain deleted of the alr gene effectively activated the TLR4-NFκB-NLRP3 pathway, triggered a robust inflammatory response, and reduced bacterial survival within host cells. Moreover, the Δalr strain exhibits lower toxicity and stronger immunogenicity in mice.

Brucella spp. in Wildlife of the Lombardy Region, Northern Italy.

Surveillance data collected in the period 2017-20 for Brucella spp. in wildlife of the Lombardy Region in northern Italy were used to describe the exposure of the wildlife species to Brucella spp. in wild boar (Sus scrofa), European brown hare (Lepus europaeus), fallow deer (Dama dama), red deer (Cervus elaphus), and roe deer (Capreolus capreolus). Among the tested species, wild boar (n=6,440) showed the highest percentage of seropositive samples (5.9%). Notably, wild boars of perifluvial area of the Po River showed higher percentages of positivity than those of the pre-Alpine district. In addition, during the hunting season in 2018, 95 organs (uterus or testes, spleen, and submandibular lymph nodes) from wild boar of the perifluvial area of the Po River were collected for bacteriological examination. Brucella suis was isolated in culture from 18.9% of tested lymph nodes. These serological and microbiological results highlight the presence of B. suis in wild boar and suggest the importance of wild boar as a reservoir for B. suis. Comparison of the spatial distribution of Brucella-seropositive wild boars with the location of backyard swine farms revealed a higher chance of contact between the two populations only in the areas where the lower percentage of seropositive samples was observed. Conversely, the high percentage of seropositive samples observed in the Po River area coupled with positive microbiological cultures suggest a greater risk of infection for the humans directly or indirectly involved in wild boar hunting activity. These results may serve as a basis to establish sound wildlife management and to adopt education campaigns aimed at reducing the risk of human infection in people involved in wild boar hunting related activities.

A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

Detection of selected pathogens in reproductive tissues of wild boars in the Campania region, southern Italy.

Monitoring disease among wildlife is critical to preserving health in both domestic animals and wildlife, and it becomes much more critical when the diseases cause significant economic damage to the livestock industry or threaten public health. Given the continuous increase in populations and its role as a reservoir for several infections, wild boar (Sus scrofa) requires special attention regarding disease surveillance and monitoring. In this study, we investigated the molecular prevalence of selected pathogens in the wild boar population of Campania, southern Italy. The prevalence of pathogens causing reproductive problems in pigs (Sus domesticus), including porcine parvovirus (PPV), porcine circovirus types 2 and 3 (PCV-2 and PCV-3), pseudorabies virus (PRV), Coxiella burnetii, and Brucella suis, was evaluated by testing the reproductive organs collected from 63 wild boars with polymerase chain reaction. The most common pathogens were PPV (44.4%) and two porcine circoviruses (14.3%). PRV and C. burnetii, on the other hand, showed a significantly lower prevalence (1.6%). No reproductive organs tested were positive for B. suis. Risk factor analysis revealed a correlation between age and PCV-2 positivity, with animals less than 12 months old having significantly higher prevalence rates.Our findings suggest that wild boars hunted in the Campania region harbour several infections potentially transmissible to other mammals' reproductive tracts. Furthermore, our results emphasized the importance of strict adherence to biosecurity protocols on domestic swine farms, especially on free-range farms, to avoid interactions between domestic and wild animals.

Lipopolysaccharides of Brucella suis S2 Impaired the Process of Decidualization in Early Pregnancy in Mice.

Brucellosis is a notorious zoonotic disease caused by Brucella, which can lead to reproductive diseases in humans and animals, such as infertility and abortion. Lipopolysaccharides (LPS) are the main virulence factor of Brucella. LPS derived from Brucella are different and non-classical and are less toxic and less active than LPS isolated from E. coli. However, the effects and possible mechanisms of Brucella LPS-caused pregnancy loss remain to be revealed. In the present study, we investigated the effects of Brucella suis S2 LPS on early pregnancy loss in mice. The results indicated that embryo implantation failure was induced by Brucella LPS treatment in a dose-dependent manner. The injection of Brucella LPS mainly resulted in fibrinolysis in the decidual area of the uterus on the 6th day post coition (dpc), infiltration of large granular cells among the decidual cells near the embryo on the 8th dpc, a large number of gaps in the decidual area, and cell necrosis around the embryo. In addition, the expression of Cyclin D3 mRNA in the uterus on the 7th and 8th dpc and IGFBP-1 mRNA and the progesterone receptor in the uterus on the 6th and 7th dpc were also inhibited. Moreover, the expression of decidualization marker Cyclin D3 and decidualization prolactin-associated protein (dPRP) in endometrial stromal cells were also inhibited by Brucella LPS treatment in vitro. In summary, Brucella LPS affect the process of endometrial decidualization in mice by affecting the structure of the decidua and the expression of decidual marker factors in endometrial stromal cells.

Genotypic peculiarities of a human brucellosis case caused by Brucella suis biovar 5.

Human brucellosis cases are rare in non-endemic countries, such as Germany, where infections are predominantly caused by Brucella melitensis. The German National Reference Laboratory for Bovine, Porcine, Ovine and Caprine Brucellosis received a suspected Brucella sp. isolate from a patient for identification. Bacteriological tests and PCR-based diagnostics showed the isolate to be B. suis, but did not yield cohesive results regarding the biovar. Whole genome sequencing and subsequent genotyping was employed for a detailed characterization of the isolate and elucidating the reason for failure of the diagnostic PCR to correctly identify the biovar. The isolate was found to be B. suis bv. 5, a rare biovar with limited geographical distribution primarily found in the Northern Caucasus. Due to a deletion in one of the target regions of the diagnostic PCR, the isolate could not be correctly typed. Based on in silico genotyping it could be excluded that the isolate was identical to one of the B. suis bv. 5 reference strains. Here, we report a rare case of a B. suis bv. 5 field isolate. Furthermore, by reporting this finding, we want to make practitioners aware of possible misinterpretation of PCR results, as it cannot be excluded that the detected deletion is common among the B. suis bv. 5 community, as there is currently a lack of field isolates.

Alr Gene in Brucella suis S2: Its Role in Lipopolysaccharide Biosynthesis and Bacterial Virulence in RAW264.7.

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain's rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella's survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.

A rapid and sensitive triplex-recombinase polymerase amplification for simultaneous differentiation of Brucella abortus, Brucella melitensi s, and Brucella suis in sera and foods.

Brucella is the causative agent of brucellosis and can be transmitted to humans through aerosolized particles or contaminated food. Brucella abortus (B. abortus), Brucella melitensis (B. melitensis), and Brucella suis (B. suis) are the most virulent of the brucellae, but the traditional detection methods to distinguish them are time-consuming and require high instrumentation. To obtain epidemiological information on Brucella during livestock slaughter and food contamination, we developed a rapid and sensitive triplex recombinant polymerase amplification (triplex-RPA) assay that can simultaneously detect and differentiate between B. abortus, B. melitensis, and B. suis. Three pairs of primers (B1O7F/B1O7R, B192F/B192R, and B285F/B285R) were designed and screened for the establishment of the triplex-RPA assay. After optimization, the assay can be completed within 20 min at 39°C with good specificity and no cross-reactivity with five common pathogens. The triplex-RPA assay has a DNA sensitivity of 1-10 pg and a minimum detection limit of 2.14 × 104-2.14 × 105 CFU g-1 in B. suis spiked samples. It is a potential tool for the detection of Brucella and can effectively differentiate between B. abortus, B. melitensis, and B. suis S2, making it a useful tool for epidemiological investigations.

The Role of Feral Swine in Human Aortic Infection With Brucella Species.

Brucellosis is one of the most common zoonotic infections in the world. Human infections are the result of direct exposure to infected animals or ingestion of unprocessed dairy products. While Brucella sp. infection has largely been eliminated from commercial cattle and swine with aggressive vaccination, there is a significant prevalence of Brucella sp. infection in the expanding population of feral swine in the US. We report the surgical treatment of a ruptured mycotic aneurysm of the abdominal aorta due to Brucella suis in a woman living in a rural community with a large population of feral swine. Vascular surgeons should be aware that brucellosis can result in arterial infection and should be considered in the differential diagnosis in patients with a history of exposure to feral swine or the ingestion of unprocessed dairy products.

Case Report: Acute Brucellosis Due to Brucella suis in a Brazilian Gold Miner Diagnosed in French Guiana.

A 29-year-old Brazilian illegal gold miner developed intermittent fever. Blood cultures were positive for Gram-negative coccobacilli and, after an initial misidentification by an automated identification system, the diagnosis of brucellosis caused by Brucella suis was confirmed. We hypothesize an association with domestic or wild swine exposure. The patient responded well to standard antibiotic therapy of brucellosis. We report the first case of human brucellosis on the Guiana Shield. This report underlines the importance of considering brucellosis in the presence of a fever of unknown origin, even in the Amazonian rainforest area, where several zoonotic diseases might be considered in the differential diagnosis of unexplained fever.

Comparison of Illumina and Oxford Nanopore Technology for genome analysis of Francisella tularensis, Bacillus anthracis, and Brucella suis.

BACKGROUND: Bacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such as Bacillus anthracis, Brucella species, and Francisella tularensis. Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each of Ba. anthracis, Br. suis and F. tularensis. Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared. RESULTS: As previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids of Ba. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades for Ba. anthracis and F. tularensis as well as multilocus sequence types for Br. suis. For F. tularensis, high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. For Ba. anthracis, only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, for Br. suis, high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions. CONCLUSIONS: In summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible for F. tularensis and Ba. anthracis, but not yet for Br. suis. The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future.

Clinical investigation and management of Brucella suis seropositive dogs: A longitudinal case series.

BACKGROUND: Brucellosis in dogs caused by Brucella suis is an emerging zoonotic disease. OBJECTIVES: To document clinical characteristics, serology, microbiology, and clinical response to treatment in B. suis-seropositive dogs. ANIMALS: Longitudinal study of 27 privately-owned dogs. Dogs that tested positive by serology, culture, or real-time polymerase chain reaction (qPCR) were included in the study. METHODS: Clinical (physical examination and imaging) and laboratory (serology, hematology, serum biochemistry, and qPCR or culture) assessments were made at baseline and after approximately 3, 6, 12, and 18 months. RESULTS: Dogs were followed for 10 895 dog days, with 17/27 dogs completing the 18-month follow-up. Ten dogs had signs consistent with brucellosis before enrollment (n = 4), at baseline (n = 2) or during follow-up (n = 6), with 2 dogs experiencing relapse of historical signs. Antibody titers persisted for the duration of follow-up in 15/17 dogs (88%). Radiographic (n = 5) and ultrasound (n = 11) findings, of variable clinical relevance, were observed. Brucella DNA and organisms were detected in 3 dogs, all of which had clinical signs, including in the milk of a bitch around the time of whelping. Brucella DNA was not detected in blood (n = 92 samples), urine (n = 80), saliva (n = 95) or preputial swabs (n = 78) at any time during follow-up. Six dogs underwent treatment, all of which achieved clinical remission although remission was not reflected by decreasing antibody titers. CONCLUSIONS AND CLINICAL IMPORTANCE: Most dogs with B. suis infections have subclinical infections. Serology is poorly associated with clinical disease. Excretion of organisms appears rare except in whelping bitches. Clinical management using antibiotics with or without surgery is recommended.

Phylogeography of Brucella suis biovar 2 with focus on Slovenian wildlife.

Brucella suis commonly infects swine but occasionally also other animal species and humans. Wild boars are the most important reservoir of B. suis biovar 2, continually infecting susceptible hosts through close contact. Nevertheless, the genetic diversity of B. suis in wildlife remains understudied. Here, we typed 17 Slovenian B. suis biovar 2 isolates obtained in 2017-2019 from wild boars (n = 16) and a hare (n = 1) using whole-genome sequencing (WGS). To assess the global phylogenetic diversity of B. suis, we compared them to 126 publicly available B. suis genomes. All Slovenian isolates fell within the biovar 2 lineage, confirming the previous multiplex PCR typing results. According to MLST-21, the wild boar isolates were of sequence types (STs) ST16 (n = 8) and ST153 (n = 8); the maximum genetic distance between isolates of the same ST was 28 wgMLST alleles. The ST153 isolates were restricted to the Slovenian-Croatian border and clustered together with the Croatian ST153 isolates from swine, indicating cross-border transmission of B. suis ST153 strain. The hare isolate was of ST40 and was genetically distant (≥ 489 alleles) from the wild boar isolates. The genome-wide phylogeny clearly separated different B. suis biovars. The present study is the first report on the population structure of B. suis in wildlife in Slovenia and shows that the Slovenian B. suis population is genetically heterogeneous. At the species level, B. suis biovars are clearly separated in the WGS-based phylogenetic tree and can therefore be reliably predicted using WGS.

Clearance of bacteria from lymph nodes in sheep immunized with Brucella suis S2 vaccine is associated with M1 macrophage activation.

Ovine brucellosis is a global zoonotic disease of sheep caused by Brucella melitensis, which inflicts a significant burden on human and animal health. Brucella suis strain S2 (B. suis S2) is a smooth live attenuated vaccine for the prevention of ovine brucellosis in China. However, no previous studies have assessed the immunogenicity of B. suis S2 vaccine after oral immunization in sheep. Here, we attempted to evaluate the ovine immune response over the course of B. suis S2 immunization and to identify in vivo predictors for vaccine development. Body temperature, serum Brucella antibodies, serum cytokines (IL-12p70 and interferon [IFN]-γ), and bacterial load in the mandibular lymph nodes (LN), superficial cervical LN, superficial inguinal LN, and spleen were investigated to determine the safety and efficacy of the vaccine. The abnormal body temperature of sheep occurred within 8 days post-infection (dpi). Brucella suis S2 persisted for a short time (< 21 dpi) in the mandibular LN. The highest level of IL-12p70 was observed at 9 dpi, whereas serum IFN-γ levels peaked at 12 dpi. Transcriptome analysis and quantitative reverse transcription PCR were performed to determine gene expression profiles in the mandibular LN of sheep. Antigen processing and presentation pathway was the dominant pathway related to the dataset. Our studies suggest that the immune response in ovine LN resembled type 1 immunity with the secretion of IL-12p70 and IFN-γ after B.suis S2 immunization and the vaccine may eliminate Brucella via stimulation of M1 macrophages through the course of Th cells.

Histopathologic and immunohistochemical findings in the placentas and fetuses of domestic swine naturally infected with Brucella suis biovar 2.

Porcine brucellosis, which is caused by Brucella suis biovar (bv) 2, is a re-emerging disease that causes reproductive problems in pigs in Europe. The pathogenesis and lesions of B. suis intrauterine infection are poorly characterized; characterization could facilitate the diagnosis and investigation of porcine brucellosis. We collected samples of placentas and fetuses for histologic and microbiologic studies during an outbreak of abortions on a pig-breeding farm in Spain. Brucella was cultured from the vaginal swabs obtained from sows that had aborted, some placentas, and fetal tissues (spleen, liver, lung, gastric content); molecular testing confirmed B. suis bv 2 infection. Histologically, there was necrotizing and hemorrhagic placentitis; suppurative hepatitis; lymphoid depletion and sinusoidal histiocytosis in the spleen, lymph nodes, and thymus; and bronchointerstitial pneumonia. Hemorrhages were observed in the umbilical cord, heart, kidneys, and brain. We detected Brucella by immunohistochemistry (IHC) in all of the placentas and fetal organs studied, specifically in the trophoblasts of the chorionic epithelium, in the cytoplasm of macrophages in the chorionic stroma, and extracellularly in necrotic debris. Furthermore, we assessed the lymphocyte population in the placentas through the use of IHC (anti-CD3, anti-Pax5 antibodies), revealing that the lymphocytic response was composed of T cells but not B cells.

Effect of ubiquitin-proteasome system and autophagy-lysosome pathway on intracellular replication of Brucella.suis.

The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are two major protein degradation pathways in eukaryotic cells. In the present study, we investigated the role of two systems and their interaction after Brucella.suis (B.suis) infected RAW264.7 murine macrophage. We demonstrated that B.suis activated ALP by upregulating LC3-Ⅱlevels as well as incomplete inhibition of P62 expression in RAW264.7 cells. On the other hand, we used pharmacological agents to confirm that ALP contributed the intracellular proliferation of B.suis. At present, the studies on the relationship between UPS and Brucella remain less understanding. In the study, we demonstrated that UPS machinery was also activated by promoting expression of 20 s proteasome after B.suis infected RAW264.7 cells, and that, the UPS could also promote intracellular proliferation of B.suis. Many recent studies propose the close link and dynamic interconversion between UPS and ALP. Currently, the experiments demonstrated that after RAW264.7 cells infected B.suis, ALP was activated following UPS inhibition, while the UPS was not effectively activated after ALP inhibition. Last, we compared the ability to promote intracellular proliferation of B.suis between UPS and ALP. The results displayed that the ability of UPS to promote intracellular proliferation of B.suis was stronger than that of ALP, and simultaneous inhibition of UPS and ALP led to seriously affection on intracellular proliferation of B.suis. All above, our research provides a better understanding on the interaction between Brucella and both systems.

Brucella suis in three dogs: presentation, diagnosis and clinical management.

Brucella suis is an emerging, zoonotic disease predominantly affecting dogs and humans that engage in feral pig hunting in Australia and other countries. Although B. suis infection in dogs shares some clinical similarities to the host-adapted species (B. canis), B. suis remains an incompletely understood pathogen in dogs with limited published data on its pathogenesis and clinical features. This case series describes the presentations, diagnosis, and clinical management of B. suis infection in three dogs: (1) a bitch with dystocia, abortion and mastitis; (2) an entire male dog with septic arthritis and presumptive osteomyelitis; and (3) a castrated male dog with lymphadenitis. Unique features of these cases are reported including the first documented detection of B. suis from milk and isolation from lymph nodes of canine patients, as well as the follow-up of pups born to a B. suis-infected bitch. Consistent with previous reports, all three dogs showed a favourable clinical response to combination antibiotic therapy with rifampicin and doxycycline. Individually tailored drug regimens were required based on the clinical presentation and other factors, including owner expectations and compliance with therapy as well as a zoonotic risk assessment (generally considered low, except around time of whelping). The authors include their recommendations for the clinical management of dogs that are at-risk or seropositive for B. suis with or without clinical signs or laboratory-confirmed infection.

Analysis of the Brucella suis Twin Arginine Translocation System and Its Substrates Shows That It Is Essential for Viability.

Bacteria use the twin arginine translocator (Tat) system to export folded proteins from the cytosol to the bacterial envelope or to the extracellular environment. As with most Gram-negative bacteria, the Tat system of the zoonotic pathogen Brucella spp. is encoded by a three-gene operon, tatABC. Our attempts, using several different strategies, to create a Brucella suis strain 1330 tat mutant were all unsuccessful. This suggested that, for B. suis, Tat is essential, in contrast to a recent report for Brucella melitensis. This was supported by our findings that two molecules that inhibit the Pseudomonas aeruginosa Tat system also inhibit B. suis, B. melitensis, and Brucella abortus growth in vitro. In a bioinformatic screen of the B. suis 1330 proteome, we identified 28 proteins with putative Tat signal sequences. We used a heterologous reporter assay based on export of the Tat-dependent amidase AmiA by using the Tat signal sequences from the Brucella proteins to confirm that 20 of the 28 candidates can engage the Tat pathway.

Immunoinformatic-guided designing of multi-epitope vaccine construct against Brucella Suis 1300.

Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to survive and multiply in a phagocytic environment and to acquire resistance under hostile conditions thus becoming a threat globally. Antibiotic resistance is posing a substantial public health threat, hence there is an unmet and urgent clinical need for immune-based non-antibiotic methods to treat brucellosis. Hence, we aimed to explore the whole proteome of Brucella suis to predict antigenic proteins as a vaccine target and designed a novel chimeric vaccine (multi-epitope vaccine) through subtractive genomics-based reverse vaccinology approaches. The applied subsequent hierarchical shortlisting resulted in the identification of Multidrug efflux Resistance-nodulation-division (RND) transporter outer membrane subunit (gene BepC) that may act as a potential vaccine target. T-cell and B-cell epitopes have been predicted from target proteins using a number of immunoinformatic methods. Six MHC I, ten MHC II, and four B-cell epitopes were used to create a 324-amino-acid MEV construct, which was coupled with appropriate linkers and adjuvant. To boost the immunological response to the vaccine, the vaccine was combined with the TLR4 agonist HBHA protein. The MEV structure predicted was found to be highly antigenic, non-toxic, non-allergenic, flexible, stable, and soluble. To confirm the interactions with the receptors, a molecular docking simulation of the MEV was done using the human TLR4 (toll-like receptor 4) and HLAs. The stability and binding of the MEV-docked complexes with TLR4 were assessed using molecular dynamics (MD) simulation. Finally, MEV was reverse translated, its cDNA structure was evaluated, and then, in silico cloning into an E. coli expression host was conducted to promote maximum vaccine protein production with appropriate post-translational modifications. These comprehensive computer calculations backed up the efficacy of the suggested MEV in protecting against B. suis infections. However, more experimental validations are needed to adequately assess the vaccine candidate's potential. HIGHLIGHTS: • Subtractive genomic analysis and reverse vaccinology for the prioritization of novel vaccine target • Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy, and structural-based studies • Molecular docking simulation method to rank based vaccine candidate and understand their binding modes.

Whole-genome sequencing (WGS) analysis of Brucella suis biovar 2 isolated from domestic pigs in Egypt for epidemiological and genetic diversity tracing.

In the current study, 14 Brucella suis biovar 2 (B. suis bv 2) strains isolated from slaughter pigs in Cairo were sequenced using Illumina technology to investigate genetic diversity, antimicrobial resistance (AMR) genes, and virulence-associated determinants. These strains were the first B. suis bv 2 isolates from Egypt. To place them in a global context, 92 genomes of B. suis were retrieved from the NCBI database and used for comparison. The in-silico analysis of MLST showed that all isolates have ST16. No resistome but 43 virulomes have been found without differences in distribution. The cgMLST classified the Egyptian B. suis strains into a complex type (CT) encompassing four distinct cgMLST sequence types. The closest relatives were strain B. suis 94/11 of an unknown origin and a Danish strain. Whole-genome sequencing analysis proved low diversity of Egyptian B. suis isolates; thus, a single introduction event is assumed. Investigation of a large number of B. suis isolates from different governorates is required to tailor control measures to avoid further spread.