Brucella BtpB Manipulates Apoptosis and Autophagic Flux in RAW264.7 Cells.

Brucella transfers effectors into host cells, manipulating cellular processes to its advantage; however, the mechanism by which effectors regulate cellular processes during infection is poorly understood. A growing number of studies have shown that apoptosis and autophagy are critical mechanisms for target cells to cope with pathogens and maintain cellular homeostasis. BtpB is a Brucella type IV secretion system effector with a complex mechanism for manipulating host infection. Here, we show that the ectopic expression of BtpB promoted DNA fragmentation. In contrast, an isogenic mutant strain, ΔbtpB, inhibited apoptosis compared to the wild-type strain B. suis S2 in RAW264.7 cells. In addition, BtpB inhibited autophagy, as determined by LC3-II protein levels, the number of LC3 puncta, and p62 degradation. We also found that BtpB reduced autophagolysosome formation and blocked the complete autophagic flux. Moreover, our results revealed that the autophagy inhibitor, chloroquine, reduces Brucella's intracellular survival. Overall, our data unveil new mechanisms of virulence implicating the effector BtpB in regulating host intracellular infection.

Investigation of the molecular characteristics of Brucella isolates from Guangxi Province, China.

BACKGROUND: Human brucellosis has become a severe public health problem in China's Guangxi Province, and there has been higher prevalence of brucellosis in this region after 2010. Both multiple locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) assay schedules were used to genotype isolates and determine relationships among isolates. RESULTS: A total of 40 isolates of Brucella were obtained from humans, pigs, and dogs from 1961 to 2016. There were at least three species of Brucella detected in Guangxi Province, Brucella melitensis, Brucella suis, and Brucella canis, with 16, 17, and 7 isolates, respectively. Of which B. suis biovar 3 was the predominant species resulting in pig brucellosis in the area examined before 2000s. Moreover, B. melitensis biovar 3 was found to be mainly responsible for human brucellosis during 2012-2016. All B. melitensis isolates in this study belonged to East Mediterranean lineage. MLVA-11 genotype 116 was the dominant genotype and represented 81.2% of the isolates. MLVA cluster analysis showed there to be 44% (7/16) brucellosis cases caused by B. melitensis with a profile of outbreak epidemic from 2012 to 2016. However, nearly 83.3% (20/24) of brucellosis cases resulting from both B. suis and B. canis showed no epidemiological links or sporadic characteristics. MLVA-16 analysis confirmed extensive genotype-sharing events between B. melitensis isolates from Guangxi and other northern provinces within China. These data revealed that there are potential epidemiology links among these strains. B. suis strains of this study showed a unique genetic lineage at the global level and may have existed historically in this area. However, present B. canis isolates were closely related to previously reported isolates in Korea, where they may have originated. MLST typing showed that the population structure of Brucella strains had changed considerably in this province; ST17 and ST21, two previously predominant populations appeared to have been replaced by recently emerging ST8 group. CONCLUSIONS: Our investigation data have inspired the hypothesis that Guangxi Province had been subject to an imported human brucellosis epidemic. Our data suggest that strains found in Northern regions of China are the principal source of infections in recent cases of human brucellosis in Guangxi Province. Comparative genomic analysis from more strains is necessary to confirm this hypothesis. This work will facilitate better understanding of the epidemiology and improve the effectiveness of control and prevention of brucellosis in this region.

Evolution and genome specialization of Brucella suis biovar 2 Iberian lineages.

BACKGROUND: Swine brucellosis caused by B. suis biovar 2 is an emergent disease in domestic pigs in Europe. The emergence of this pathogen has been linked to the increase of extensive pig farms and the high density of infected wild boars (Sus scrofa). In Portugal and Spain, the majority of strains share specific molecular characteristics, which allowed establishing an Iberian clonal lineage. However, several strains isolated from wild boars in the North-East region of Spain are similar to strains isolated in different Central European countries. RESULTS: Comparative analysis of five newly fully sequenced B. suis biovar 2 strains belonging to the main circulating clones in Iberian Peninsula, with publicly available Brucella spp. genomes, revealed that strains from Iberian clonal lineage share 74% similarity with those reference genomes. Besides the 210 kb translocation event present in all biovar 2 strains, an inversion with 944 kb was presented in chromosome I of strains from the Iberian clone. At left and right crossover points, the inversion disrupted a TRAP dicarboxylate transporter, DctM subunit, and an integral membrane protein TerC. The gene dctM is well conserved in Brucella spp. except in strains from the Iberian clonal lineage. Intraspecies comparative analysis also exposed a number of biovar-, haplotype- and strain-specific insertion-deletion (INDELs) events and single nucleotide polymorphisms (SNPs) that could explain differences in virulence and host specificities. Most discriminative mutations were associated to membrane related molecules (29%) and enzymes involved in catabolism processes (20%). Molecular identification of both B. suis biovar 2 clonal lineages could be easily achieved using the target-PCR procedures established in this work for the evaluated INDELs. CONCLUSION: Whole-genome analyses supports that the B. suis biovar 2 Iberian clonal lineage evolved from the Central-European lineage and suggests that the genomic specialization of this pathogen in the Iberian Peninsula is independent of a specific genomic event(s), but instead driven by allopatric speciation, resulting in the establishment of a new ecovar.

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis.

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the 'Bruce ladder' multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.

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.

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.

A designed peptide-based vaccine to combat Brucella melitensis, B. suis and B. abortus: Harnessing an epitope mapping and immunoinformatics approach.

Vaccines against Brucella abortus, B. melitensis and B. suis have been based on weakened or killed bacteria, however there is no recombinant vaccine for disease prevention or therapy. This study attempted to predict IFN-γ epitopes, T cell cytotoxicity, and T lymphocytes in order to produce a multiepitope vaccine based on BtpA, Omp16, Omp28, virB10, Omp25, and Omp31 antigens against B. melitensis, B. abortus, and B. suis. AAY, GPGPG, and EAAAK peptides were used as epitope linkers, while the PADRE sequence was used as a Toll-like receptor 2 (TLR2) and TLR4 agonist. The final construct included 389 amino acids, and was a soluble protein with a molecular weight of 41.3 kDa, and nonallergenic and antigenic properties. Based on molecular docking studies, molecular dynamics simulations such as Gyration, RMSF, and RMSD, as well as tertiary structure validation methods, the modeled protein had a stable structure capable of interacting with TLR2/4. As a result, this novel vaccine may stimulate immune responses in B and T cells, and could prevent infection by B. suis, B. abortus, and B. melitensis.

Machine Learning Algorithms for Classification of MALDI-TOF MS Spectra from Phylogenetically Closely Related Species Brucella melitensis, Brucella abortus and Brucella suis.

(1) Background: MALDI-TOF mass spectrometry (MS) is the gold standard for microbial fingerprinting, however, for phylogenetically closely related species, the resolution power drops down to the genus level. In this study, we analyzed MALDI-TOF spectra from 44 strains of B. melitensis, B. suis and B. abortus to identify the optimal classification method within popular supervised and unsupervised machine learning (ML) algorithms. (2) Methods: A consensus feature selection strategy was applied to pinpoint from among the 500 MS features those that yielded the best ML model and that may play a role in species differentiation. Unsupervised k-means and hierarchical agglomerative clustering were evaluated using the silhouette coefficient, while the supervised classifiers Random Forest, Support Vector Machine, Neural Network, and Multinomial Logistic Regression were explored in a fine-tuning manner using nested k-fold cross validation (CV) with a feature reduction step between the two CV loops. (3) Results: Sixteen differentially expressed peaks were identified and used to feed ML classifiers. Unsupervised and optimized supervised models displayed excellent predictive performances with 100% accuracy. The suitability of the consensus feature selection strategy for learning system accuracy was shown. (4) Conclusion: A meaningful ML approach is here introduced, to enhance Brucella spp. classification using MALDI-TOF MS data.

RNA-Seq Analysis Reveals the Role of Omp16 in Brucella-Infected RAW264.7 Cells.

Brucellosis is an endemic zoonotic infectious disease in the majority of developing countries, which causes huge economic losses. As immunogenic and protective antigens at the surface of Brucella spp., outer membrane proteins (Omps) are particularly attractive for developing vaccine and could have more relevant role in host-pathogen interactions. Omp16, a homolog to peptidoglycan-associated lipoproteins (Pals), is essential for Brucella survival in vitro. At present, the functions of Omp16 have been poorly studied. Here, the gene expression profile of RAW264.7 cells infected with Brucella suis vaccine strain 2 (B. suis S2) and ΔOmp16 was analyzed by RNA-seq to investigate the cellular response immediately after Brucella entry. The RNA-sequence analysis revealed that a total of 303 genes were significantly regulated by B. suis S2 24 h post-infection. Of these, 273 differentially expressed genes (DEGs) were upregulated, and 30 DEGs were downregulated. These DEGs were mainly involved in innate immune signaling pathways, including pattern recognition receptors (PRRs), proinflammatory cytokines, and chemokines by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In ΔOmp16-infected cells, the expression of 52 total cells genes was significantly upregulated and that of 9 total cells genes were downregulated compared to B. suis S2-infected RAW264.7 cells. The KEGG pathway analysis showed that several upregulated genes were proinflammatory cytokines and chemokines, such as interleukin (IL)-6, IL-11, IL-12ß, C-C motif chemokine (CCL2), and CCL22. All together, we clearly demonstrate that ΔOmp16 can alter macrophage immune-related pathways to increase proinflammatory cytokines and chemokines, which provide insights into illuminating the Brucella pathogenic strategies.

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.

Seroprevalence and Molecular Identification of Brucella spp. in Camels in Egypt.

Brucellosis is one of the most important worldwide zoonoses of many countries including Egypt. Camel brucellosis has not gained much attention in Egypt yet. This study is focused on the three governorates with the highest camel populations and the largest camel markets in the country to determine the disease seroprevalence and identify the Brucella species in local camel holdings. In total, 381 serum samples were collected from male and female camels from Giza, Aswan, and Al-Bahr Al-Ahmar (the Red Sea) governorates. Samples were serologically examined using the Rose-Bengal plate test (RBPT), indirect ELISA (i-ELISA), competitive ELISA (c-ELISA) and complement fixation test (CFT). Brucella antibodies were detected in 59 (15.5%), 87 (22.8%), 77 (20.2%) and 118 (31.0%) of sera by RBPT, i-ELISA, c-ELISA and CFT, respectively. Using real-time PCR, Brucella DNA was amplified in 32 (8.4%) seropositive samples including Brucella abortus (25/32), Brucella suis (5/32) and Brucella melitensis (2/32), defining a complex epidemiological status. To the best of our knowledge, this is the first study reporting Brucella suis DNA in camel serum. The risk-associated factors including age, sex, breed and geographical distribution were statistically analyzed, showing non-significant association with seroprevalence. The results of this study will raise awareness for camel brucellosis and help develop effective control strategies.

Monitoring results of wild boar (Sus scrofa) in The Netherlands: analyses of serological results and the first identification of Brucella suis biovar 2.

In Europe, wild boar populations pose an increasing risk for livestock and humans due to the transmission of animal and zoonotic infectious diseases, such as African swine fever and brucellosis. Brucella suis is widespread among wild boar in many European countries. In The Netherlands the prevalence of B. suis among wild boar has not been investigated so far, despite the high number of pig farms and the growing wild boar population. The Netherlands has a Brucella-free status for the livestock species. The objective of this study is to investigate the presence and distribution of B. suis in wild boars in The Netherlands and to assess the value of the different laboratory tests available for testing wild boars. A total of 2057 sera and 180 tonsils of wild boar were collected between 2010 and 2015. The sera were tested for Brucella antibodies and the tonsils were tested for Brucella spp. B. suis biovar 2 was detected by MLVA/MLST and culture in wild boar from the province of Limburg, while seropositive wild boar were obtained from the provinces of Limburg, Noord Brabant and Gelderland suggesting the northwards spread of B. suis biovar 2. In this paper, we describe the first isolation of B. suis biovar 2 in wild boar in The Netherlands.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infection with Brucella abortus, B. melitensis and B. suis.

The infection with Brucella abortus, Brucella melitensis and Brucella suis has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on the eligibility of the infection with B. abortus, B. melitensis and B. suis to be listed, Article 9 for the categorisation of the infection with B. abortus, B. melitensis and B. suis according to disease prevention and control rules as in Annex IV and Article 8 on the list of animal species related to the infection with B. abortus, B. melitensis and B. suis. The assessment has been performed following a methodology composed of information collection and compilation, expert judgement on each criterion at individual and, if no consensus was reached before, also at collective level. The output is composed of the categorical answer, and for the questions where no consensus was reached, the different supporting views are reported. Details on the methodology used for this assessment are explained in a separate opinion. According to the assessment performed, the infection with B. abortus, B. melitensis and B. suis can be considered eligible to be listed for Union intervention as laid down in Article 5(3) of the AHL. The disease complies with the criteria as in Sections 2, 3, 4 and 5 of Annex IV of the AHL, for the application of the disease prevention and control rules referred to in points (b), (c), (d) and (e) of Article 9(1). The animal species to be listed for the infection with B. abortus, B. melitensis and B. suis according to Article 8(3) criteria are several mammal species, as indicated in the present opinion.

A genome-wide SNP-based phylogenetic analysis distinguishes different biovars of Brucella suis.

Brucellosis is an important zoonotic disease caused by Brucella spp. Brucella suis is the etiological agent of porcine brucellosis. B. suis is the most genetically diverged species within the genus Brucella. We present the first large-scale B. suis phylogenetic analysis based on an alignment-free k-mer approach of gathering polymorphic sites from whole genome sequences. Genome-wide core-SNP based phylogenetic tree clearly differentiated and discriminated the B. suis biovars and the vaccine strain into different clades. A total of 16,756 SNPs were identified from the genome sequences of 54 B. suis strains. Also, biovar-specific SNPs were identified. The vaccine strain B. suis S2-30 is extensively used in China, which was discriminated from all biovars with the accumulation of the highest number of SNPs. We have also identified the SNPs between B. suis vaccine strain S2-30 and its closest homolog, B. suis biovar 513UK. The highest number of mutations (22) was observed in the phosphomannomutase (pmm) gene essential for the synthesis of O-antigen. Also, mutations were identified in several virulent genes including genes coding for type IV secretion system and the effector proteins, which could be responsible for the attenuated virulence of B. suis S2-30.

The Complete Genome of Brucella Suis 019 Provides Insights on Cross-Species Infection.

Brucella species are the most important zoonotic pathogens worldwide and cause considerable harm to humans and animals. In this study, we presented the complete genome of B. suis 019 isolated from sheep (ovine) with epididymitis. B. suis 019 has a rough phenotype and can infect sheep, rhesus monkeys and possibly humans. The comparative genome analysis demonstrated that B. suis 019 is closest to the vaccine strain B. suis bv. 1 str. S2. Further analysis associated the rsh gene to the pathogenicity of B. suis 019, and the WbkA gene to the rough phenotype of B. suis 019. The 019 complete genome data was deposited in the GenBank database with ID PRJNA308608.

Brucella suis Vaccine Strain 2 Induces Endoplasmic Reticulum Stress that Affects Intracellular Replication in Goat Trophoblast Cells In vitro.

Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection.