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      Molecular Characteristics of Brucella Isolates Collected From Humans in Hainan Province, China

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          Abstract

          Brucellosis has been reported in several regions of Hainan Province, but the extent of the disease has not been fully elucidated. Conventional biotyping methods, multiple locus variable number tandem repeats analysis (MLVA), and single-nucleotide polymorphisms (SNPs) from draft genome sequencing were employed to characterize the strains. There were four biovars ( Brucella melitensis bv. 1, 2, and 3 and Brucella suis bv. 3) detected, which showed that the biovar diversity of Brucella in Hainan is higher than in other areas of China. Both B. melitensis bv. 3 and B. suis bv. 3 were dominant species and showed epidemiology patterns that were compatible with both southern and northern China. Eight of MLVA-11 genotypes were known (31, 111, 116, 120, 136, 291, 297, and 345), and the remaining seven were novel (HN11-1 to HN11-7); these data showed that Brucella strains in this study had multiple geographic origins and exhibited characteristics of origin and evolution of co-existing imported and Hainan specific lineage. A total of 41 strains were found, belonging to 37 unique genotypes that each represented a single strain, which suggests that these strains were not directly related epidemiologically and indicates that the epidemic characteristics of human brucellosis in Hainan was dominated by sporadic strains. The high HGDI values were observed in MLVA-8, MLVA-11, and MLVA-16 among two species, suggesting considerable genetic diversity among these species. MST is characterized based on MLVA-16 that was found both throughout China and on a global level and showed that strains of this study had significant genetic differences with strains from many parts of the globe and seemingly represent a unique genetic lineage. Whole-genome SNP analysis showed that four B. melitensis were closely related to strains from China’s northern provinces, and the source of infection was partly of human brucellosis in this province that may have been from these regions. The B. suis were closely related to strains from the United States, and further investigation of the transportation of animals, such as pigs, is needed to elucidate the origins of these strains.

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          PHYLOViZ 2.0: providing scalable data integration and visualization for multiple phylogenetic inference methods.

          High Throughput Sequencing provides a cost effective means of generating high resolution data for hundreds or even thousands of strains, and is rapidly superseding methodologies based on a few genomic loci. The wealth of genomic data deposited on public databases such as Sequence Read Archive/European Nucleotide Archive provides a powerful resource for evolutionary analysis and epidemiological surveillance. However, many of the analysis tools currently available do not scale well to these large datasets, nor provide the means to fully integrate ancillary data. Here we present PHYLOViZ 2.0, an extension of PHYLOViZ tool, a platform independent Java tool that allows phylogenetic inference and data visualization for large datasets of sequence based typing methods, including Single Nucleotide Polymorphism (SNP) and whole genome/core genome Multilocus Sequence Typing (wg/cgMLST) analysis. PHYLOViZ 2.0 incorporates new data analysis algorithms and new visualization modules, as well as the capability of saving projects for subsequent work or for dissemination of results.
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            Pathogenesis and immunobiology of brucellosis: review of Brucella-host interactions.

            This review of Brucella-host interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system-dependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of next-generation sequences of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent targets for improved, safer brucellosis vaccines and therapeutics.
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              Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay

              Background The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification. Results Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2). Conclusion The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                27 March 2020
                2020
                : 11
                : 452
                Affiliations
                [1] 1State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention , Beijing, China
                [2] 2Hainan Provincial People’s Hospital , Haikou, China
                [3] 3Sanya People’s Hospital , Sanya, China
                [4] 4Ulanqab Centre for Endemic Disease Prevention and Control , Jining, China
                [5] 5Inner Mongolia Autonomous Region Center for Comprehensive Disease Control and Prevention , Huhhot, China
                Author notes

                Edited by: Narjol González-Escalona, United States Food and Drug Administration, United States

                Reviewed by: David O’Callaghan, Université de Montpellier, France; Luca Freddi, Agence Nationale de Sécurité Sanitaire de l’Alimentation, de l’Environnement et du Travail (ANSES), France; Ana Cristina Ferreira, National Institute of Agricultural and Veterinary Research (INIAV), Portugal

                *Correspondence: Zhi Guo Liu, liuzhiguo@ 123456icdc.cn ; wlcblzg@ 123456126.com

                These authors have contributed equally to this work

                This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2020.00452
                7120952
                32292391
                60c259a3-b541-4cda-9a21-47c6df300010
                Copyright © 2020 Li, Wang, Zhu, Wang, Cheng, Li and Liu.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 13 November 2019
                : 02 March 2020
                Page count
                Figures: 9, Tables: 2, Equations: 0, References: 40, Pages: 14, Words: 0
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                brucella melitensis,brucella suis,multiple loci variable number tandem repeats analysis,single-nucleotide polymorphism,hainan

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