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.

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.

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.

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.

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.

Detection of Brucella S2 vaccine strain by a loop-mediated isothermal amplification (LAMP) method.

Introduction: Brucellosis is a highly prevalent zoonotic disease caused by Brucella spp. Brucella suis S2 vaccination is an effective strategy to prevent animal brucellosis. However, S2 induces antibodies against the smooth lipopolysaccharide,making it challenging to distinguish field infected from vaccinated livestock. Early and accurate diagnosis is essential for infection control and prevention. In this study, we aimed to develop a quick and accurate assay to distinguish the BrucellaS2 vaccine strain from closely related B. abortus and B. melitensis. Methods: Whole-genome sequencing of B. suis S2 was performed, and the sequence was compared with that of the genomes of B. abortus and B. melitensis. One specific gene, GL_0002189, was selected as a marker to differentiate the BrucellaS2vaccine strain from B. abortus and B. melitensis. A loop-mediated isothermal amplification (LAMP) assay was developed, based on the GL_0002189 gene, and then assessed for target specificity, lower limit of detection, and repeatability. Results: Our results revealed that there was no cross-reaction with other strains, and the LAMP assay displayed high sensitivity for detecting S2 with a minimum detection limit of 18.9×103 copies/µL DNA input, it is nearly 100 times higher than conventional PCR technology. Concordance between the LAMP assay and a conventional polymerase chain reaction method was assessed using 54 blood samples collected from sheep with suspected brucellosis. Total concordance between the two assays was 92.6%, without a significant difference (p > 0.05) in the test results. Conclusion: This is the first report of a LAMP assay for the detection of the B. suis S2vaccine strain. Our approach can be helpful for the control and eradication of brucellosis, and its simplicity in requiring no specialized equipment or personnel makes it useful for implementation in resource-limited settings as well as for field use.

Isolation and molecular confirmation of Brucella suis biovar 2 from slaughtered pigs: an unanticipated biovar from domestic pigs in Egypt.

BACKGROUND: Brucella suis is a zoonotic pathogen with a serious impact on public health and the pig industry worldwide. Information regarding B. suis in pigs in Egypt is scarce. This study aimed to investigate the prevalence of B. suis in slaughtered domestic pigs at El-Basatin abattoir in Cairo, Egypt. A total of 1,116 domestic pigs slaughtered in 2020 were sampled for Brucella isolation and identification. Identified Brucella isolates were molecularly confirmed at species, and biovar levels using Bruce ladder PCR and Suis ladder multiplex PCR. Additionally, high-risk practices of 16 abattoir workers (4 veterinarians, 10 butchering and evisceration workers, and 2 scalding workers) were investigated using a pre-piloted structured questionnaire. RESULTS: Brucella isolates were recovered from 1.3% of examined pigs (n = 14) at consistently low rates (1.1-2.9%) across the year of sampling from February to December 2020. All isolates were confirmed as B. suis biovar (bv) 2. Remarkably, 92.9% (13/14) of isolates showed atypical ability to produce H2S and hence were considered as B. suis bv2 atypical phenotype. The prevalence was higher in males (1.8%) than in females (0.9). However, this difference was not significant (Odds ratio = 1.9; CI 95% 0.7 - 5.7; P = 0.2). No detectable pathological lesions were associated with B. suis bv2 infection in examined pigs. All strains were isolated from cervical lymph nodes, highlighting a potential oral transmission. High-risk practices were recorded among swine abattoir workers in this study: 75% do not wear gloves or disinfect their knives daily, and 18.8% were willing to work with open wound injuries. CONCLUSIONS: To the best of our knowledge, this is the first isolation of B. suis bv2 in Egypt. Detection of H2S producing B. suis bv2 atypical phenotype is alarming as it may result in misinterpretation of these isolates as highly human pathogenic B. suis bv1 in Egypt and possibly elsewhere. Further epidemiological tracing studies are crucial for the detection of the origin of this biovar. Including pigs in the national surveillance program of brucellosis, and an education program for swine abattoir workers about occupational risk of B. suis is a need in Egypt.

A LysR Transcriptional Regulator Manipulates Macrophage Autophagy Flux During Brucella Infection.

Brucella, the intracellular bacteria, have evolved subtle strategies to efficiently survive and replicate in macrophages. However, the virulence effector proteins involved are still unclear. LysR-type transcriptional regulators (lttrs) are the largest regulator family with diverse function in prokaryotes. However, very little is known about the role of LysR regulators in the Brucella spp. Here, a BSS2_II0858 gene, encoded as one of the LysR-type regulators, was studied. We successfully constructed a BSS2_II0858 deletion mutant, Δ0858, and complementation strain CΔ0858 in Brucella suis S2. The cell apoptosis induced by B. suis S2 and its derivatives were detected by flow cytometry. The autophagy was then assessed by immunoblot analysis using the IL3I/II and p62 makers. In addition, the autophagy flux was evaluated by double fluorescent labeling method for autophagy marker protein LC3. Our studies demonstrated that B. suis S2 and its derivatives inhibited the programmed cell death in early stage and promoted apoptosis in the later stage during infection in RAW264.7 cells. The BSS2_II0858 gene was found to play no role during apoptosis according to these results. Compared with the wild-type strain, Δ0858 mutant can stimulate the conversion of LC3-I to LC3-II and markedly inhibited the autophagy flux at early stage leading to obvious autophagosome accumulation. This study explored the function of BSS2_II0858 gene and may provide new insights for understanding the mechanisms involved in the survival of Brucella in macrophages.

Phenotypic and molecular identification of Brucella suis biotype 1 in a pig from Brazil-case report.

Brucellosis is a zoonotic disease with a global impact. Brucella suis is one of the most pathogenic species to humans, requiring different measures for the control and/or eradication of the disease. The serological investigation for brucellosis was performed in pigs, horses, dogs, and cattle on a farm with a history of abortion in sows and necropsy of a boar with severe necrosuppurative orchitis. One sow, two cows, and two dogs reveled positive to Rose Bengal Test (RBT), although only the sow had a confirmatory outcome in 2-mercaptoethanol (2-ME). The 2-ME-positive sow was euthanized and microbiological culture of lymph nodes and liver followed by biochemical characterization allowed phenotypic characterization of Brucella suis biotype 1. PCR multiplex Bruce-ladder and Suis-ladder enabled molecular confirmation, respectively, of Brucella suis and biotype 1. The transmission aspects of B. suis to pigs and other domestic species, the combination of diagnostic procedures to diagnosis, as well as human health concerns of brucellosis are discussed.

The Use of Flocked Swabs with a Protective Medium Increases the Recovery of Live Brucella spp. and DNA Detection.

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.

Adhesive Functions or Pseudogenization of Type Va Autotransporters in Brucella Species.

Adhesion to host cells is a key step for successful infection of many bacterial pathogens and may define tropism to different host tissues. To do so, bacteria display adhesins on their surfaces. Brucella is an intracellular pathogen capable of proliferating in a wide variety of cell types. It has been described that BmaC, a large protein that belongs to the classical (type Va) autotransporter family, is required for efficient adhesion of Brucella suis strain 1330 to epithelial cells and fibronectin. Here we show that B. suis 1330 harbors two other type Va autotransporters (BmaA and BmaB), which, although much smaller, share significant sequence similarities with BmaC and contain the essential domains to mediate proper protein translocation to the bacterial surface. Gain and loss of function studies indicated that BmaA, BmaB, and BmaC contribute, to a greater or lesser degree, to adhesion of B. suis 1330 to different cells such as synovial fibroblasts, osteoblasts, trophoblasts, and polarized epithelial cells as well as to extracellular matrix components. It was previously shown that BmaC localizes to a single bacterial pole. Interestingly, we observed here that, similar to BmaC, the BmaB adhesin is localized mostly at a single cell pole, reinforcing the hypothesis that Brucella displays an adhesive pole. Although Brucella species have strikingly similar genomes, they clearly differ in their host preferences. Mainly, the differences identified between species appear to be at loci encoding surface proteins. A careful in silico analysis of the putative type Va autotransporter orthologues from several Brucella strains showed that the bmaB locus from Brucella abortus and both, the bmaA and bmaC loci from Brucella melitensis are pseudogenes in all strains analyzed. Results reported here evidence that all three autotransporters play a role in the adhesion properties of B. suis 1330. However, Brucella spp. exhibit extensive variations in the repertoire of functional adhesins of the classical autotransporter family that can be displayed on the bacterial surface, making them an interesting target for future studies on host preference and tropism.

Genomic insights into Brucella.

Brucellosis is a zoonotic disease caused by certain species of Brucella. Each species has its preferred host animal, though it can infect other animals too. For a longer period, only six classical species were recognized in the genus Brucella. No vaccine is available for human brucellosis. Therefore, human brucellosis can be controlled only by controlling brucellosis in animals. The genus is now expanding with the newly isolated atypical strains from various animals, including marine mammals. Presently, 12 species of Brucella have been recognized. The first genome of Brucella was released in 2002, and today, we have more than 1500 genomes of Brucella spp. isolated worldwide. Multiple genome sequences are available for the major zoonotic species, B. abortus, B. melitensis, and B. suis. The Brucella genome has two chromosomes with the approximate sizes of 2.1 and 1.2 Mbp. The genome of Brucella is highly conserved across all the species at the nucleotide level. One of the unanswered questions is what makes host preference in different species of Brucella. Here, I summarize the recent advancements in the Brucella genomics research.

Omp16, a conserved peptidoglycan-associated lipoprotein, is involved in Brucella virulence in vitro.

Brucella, the bacterial agent of common zoonotic brucellosis, primarily infects specific animal species. The Brucella outer membrane proteins (Omps) are particularly attractive for developing vaccine and improving diagnostic tests and are associated with the virulence of smooth Brucella strains. Omp16 is a homologue to peptidoglycan-associated lipoproteins (Pals), and an omp16 mutant has not been generated in any Brucella strain until now. Very little is known about the functions and pathogenic mechanisms of Omp16 in Brucella. Here, we confirmed that Omp16 has a conserved Pal domain and is highly conserved in Brucella. We attempted to delete omp16 in Brucella suis vaccine strain 2 (B. suis S2) without success, which shows that Omp16 is vital for Brucella survival. We acquired a B. suis S2 Omp16 mutant via conditional complementation. Omp16 deficiency impaired Brucella outer membrane integrity and activity in vitro. Moreover, inactivation of Omp16 decreased bacterial intracellular survival in macrophage RAW 264.7 cells. B. suis S2 and its derivatives induced marked expression of IL-1ß, IL-6, and TNF-a mRNA in Raw 264.7 cells. Whereas inactivation of Omp16 in Brucella enhanced IL-1ß and IL-6 expression in Raw 264.7 cells. Altogether, these findings show that the Brucella Omp16 mutant was obtained via conditional complementation and confirmed that Omp16 can maintain outer membrane integrity and be involved in bacterial virulence in Brucella in vitro and in vivo. These results will be important in uncovering the pathogenic mechanisms of Brucella.

First isolation and characterization of Brucella suis from yak.

Brucella spp., facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonosis, affect public health and safety. A Brucella strain was isolated from yak in Qinghai Province. To detect whether this isolate could cause an outbreak of brucellosis and to reveal its genetic characteristics, several typing and whole-genome sequencing methods were applied to identify its species and genetic characteristics. Phylogenetic analysis based on MLVA and whole-genome sequencing revealed the genetic characteristics of the isolated strain. The results showed that the isolated strain is a B. suis biovar 1 smooth strain, and this isolate was named B. suis QH05. The results of comparative genomics and MLVA showed that B. suis QH05 is not a vaccine strain. Comparison with other B. suis strains isolated from humans and animals indicated that B. suis QH05 may be linked to specific animal and human sources. In conclusion, B. suis QH05 does not belong to the Brucella epidemic species in China, and as the first isolation of B. suis from yak, this strain expands the host range of B. suis.

Phylogeography and epidemiology of Brucella suis biovar 2 in wildlife and domestic swine.

Swine brucellosis due to Brucella suis biovar 2 (bv2) is enzootic in wild boar and hare in continental Europe and may cause major economic losses to the pig industry, mainly in free-ranged pig farms. The high nucleotide identity found among the B. suis biovar 2 isolates has long hindered the full understanding of the epidemiology and the phylogeography of the disease. Here, we used multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) and whole-genome analysis to identify single-nucleotide polymorphisms (SNPs) in order to gain insights from the largest B. suis bv2 dataset analyzed so far composed of domestic pigs and wildlife isolates collected throughout Europe since the 1970s. We found four major clades with a specific phylogeographic pattern. The Iberian clade contains isolates exclusively from the Iberian Peninsula. The Central European clade includes most isolates from France, Northern Italy, Switzerland and an important proportion of those of Northern Spain. The Eastern European clade clustered isolates from Croatia and Hungary mainly but also from areas of France, Germany, Italy and Poland. Finally, a separated Sardinian clade grouped three isolates from this island. At fine scale, MLVA demonstrated an endemic status of the infection in Europe and it allowed tracking a large outbreak formed by different farms from Spain linked to the same infection source. The whole genome SNP analysis showed that the strains form genetically distinct clades, shared between wild boar and pigs, in agreement with the MLVA clades. Interestingly, all hare isolates clustered together within two groups composed exclusively of wildlife isolates. Our results support the hypothesis that maintenance and spread of B. suis bv2 in Europe is a dynamic process linked to the natural expansion of wild boar as the main wild reservoir of the infection, while spread over long distances is found largely dependent on anthropogenic activities.