Bovine tuberculosis (bTB) is an endemic zoonosis significantly affects animal health in Burkina Faso. The primary causative agent is Mycobacterium tuberculosis (M. tuberculosis) complex, mainly M. bovis. Cattle are co...Bovine tuberculosis (bTB) is an endemic zoonosis significantly affects animal health in Burkina Faso. The primary causative agent is Mycobacterium tuberculosis (M. tuberculosis) complex, mainly M. bovis. Cattle are considered as natural reservoir of M. bovis. However, in Burkina Faso, the circulation of these strains remains poorly understood and documented. This study aimed to identify and characterize Mycobacterium strains from suspected carcasses during routine meat inspection at Bobo-Dioulasso refrigerated slaughterhouse. A prospective cross-sectional study was conducted from January 2021 to December 2022 on cases of seizures linked to suspected bovine tuberculosis. Microbiological and molecular analyzes were used for mycobacterial strain isolation and characterization. Out of 50 samples, 24% tested positive by microscopy and 12% by culture. Molecular analysis identified 6 strains of Mycobacteria, exclusively Mycobacterium bovis specifically the subspecies bovis (Mycobacterium bovis subsp bovis). In conclusion, M. bovis subsp bovis is the primary agent responsible for bovine tuberculosis in Bobo-Dioulasso. Continuous monitoring of mycobacterial strains is therefore necessary for the effective control of this pathology in the local cattle population.展开更多
Objective To identify the novel species ‘Mycobacterium fukienense＇ sp. nov of Mycobacterium chelonoe/abscessus complex from tuberculosis patients in Fujian Province, China. Methods Five of 27 clinical Mycobucterium ...Objective To identify the novel species ‘Mycobacterium fukienense＇ sp. nov of Mycobacterium chelonoe/abscessus complex from tuberculosis patients in Fujian Province, China. Methods Five of 27 clinical Mycobucterium isolates （CIs） were previously identified as M. chelonoe/obscessus complex by sequencing the hsp65, rpoB, 165-235 rRNA internal transcribed spacer region （its）, recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobocterium. Clinical Mycobecterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. Results The 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 165-235 rRNA internal tronscribed spacer region （its）, sodA, and recA genes as compared with the M. obscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. Conclusion The novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. cheloneo/abscessus complex.展开更多
Objective:To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E.coli) under optimistic conditions,obtain and identify protein expressed,analyze the structure and characteristics of the...Objective:To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E.coli) under optimistic conditions,obtain and identify protein expressed,analyze the structure and characteristics of the protein using bioinformatics methods for future applications.Methods: Rv3265c gene from Mycobacterium tuberculosis H37Rv was amplified by polymerase chain reaction,and was cloned into the pET-30a vector after purification and recovery.The recombinant plasmid was sequenced and expressed in E.coli BL21(DE3),and then purified and identified by western blotting.The essential physical-chemical properties of the protein were predicated by bioinformatics tools,including subcellular location,secondary structure,domains,antigenic epitopes,etc.Tertiary structure of the protein based on homology modeling was estabUshed,while multi-sequence homological alignment and phylogenetic analysis were preformed.Results:The recombinant protein was obtained in soluble fraction from expression system in E.coli B121(DE3) carrying pET30- Rv3265c plasmid,and Rv3265c gene was expressed correctly.Bioinformatics analysis showed the protein contained no signal peptide and transmembrane helices,located outside of membrane.Secondary structure analysis revealed it containedα-helix,extended strand and random coil,46.8%,14.6%,38.6%,respectively.Furthermore,it possessed six potential antigenic epitopes,one glycosyl transferase domain.A simple three-dimensional model of this protein was constructed by Swiss-model sever.Both sequences and structures were conservative and especial either in gene or in protein.Conclusions:Rv3265c gene might be a desirable molecular target for anti-tuberculosis drug and vaccine.The purified protein from expression will be utilized to study the kinetics of L-rhamnosyltransferase and to develope an enzyme assay for screening vaccine or drug.展开更多
Objective:To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis（MTU）,express the encoded fusing protein in Escherichia coli（E.coli）,identify protein acquired,and predict the structure and function of the p...Objective:To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis（MTU）,express the encoded fusing protein in Escherichia coli（E.coli）,identify protein acquired,and predict the structure and function of the protein utilizing methods of bioinformatics.Methods:fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR.The fbpB-esxA fusing gene Iigated by（Gly<sub>4</sub>Ser）<sub>3</sub> linker was gained by means of Gene Splicing by Overlapping Extension PCU（SOEPCR）, and fusing gene was cloned into expression vector pET-30a.The recombinant plasmid was sequenced and expressed in E.coli BL21（DE3）.The protein was identified by Western blot using anti-HIS antibody.Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics.Results:The UNA sequences fbpB-esxA were identical with that published by GenBank.The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E.coli B1.21（DE3） under the induction of IPTG.Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes.Conclusions:The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag.Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly.The existence of linker doesn’t affect immunogenicity of Ag85B and ESAT-6.It will allow lor characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent.展开更多
Objectives To evaluate the immunogenicity of Mycobacterium intracellulare proteins and determine the cross-reactive proteins between M.intracellulare and M.tuberculosis.Methods Protein extracts from M.intracellulare w...Objectives To evaluate the immunogenicity of Mycobacterium intracellulare proteins and determine the cross-reactive proteins between M.intracellulare and M.tuberculosis.Methods Protein extracts from M.intracellulare were used to immunize BALB/c mice.The antigens were evaluated using cellular and humoral immunoassays.The common genes between M.intracellular and M.tuberculosis were identified using genome-wide comparative analysis,and cross-reactive proteins were screened using immunoproteome microarrays.Results Immunization with M.intracellulare proteins induced significantly higher levels of the cytokines interferon-γ(IFN-γ),interleukin-2(IL-2),interleukin-12(IL-12),interleukin-6(IL-6)and immunoglobulins IgG,IgG1,IgM,and IgG2a in mouse serum.Bone marrow-derived macrophages isolated from mice immunized with M.intracellulare antigens displayed significantly lower bacillary loads than those isolated from mice immunized with adjuvants.Whole-genome sequence analysis revealed 396 common genes between M.intracellulare and M.tuberculosis.Microchip hybridization with M.tuberculosis proteins revealed the presence of 478 proteins in the serum of mice immunized with M.intracellulare protein extracts.Sixty common antigens were found using both microchip and genomic comparative analyses.Conclusion This is the advanced study to investigate the immunogenicity of M.intracellulare proteins and the cross-reactive proteins between M.intracellulare and M.tuberculosis.The results revealed the presence of a number of cross-reactive proteins between M.intracellulare and M.tuberculosis.Therefore,this study provides a new way of identifying immunogenic proteins for use in tuberculosis vaccines against both M.intracellulare and M.tuberculosis in future.展开更多
Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23...Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23S rRNA gene internal transcribed spacer sequence for the genotyping of mycobacteria to the species level. A pair of primers and a set of genus- and species-specific probes were designed from the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer, and 23S rRNA gene sequences of mycobacteria. We used a novel multiplex probe array for identification of 266 clinical specimens obtained from patients with mycobaterial infection. The results showed that the overall specificity and sensitivity of our novel probe array were both 100% for the genus-specific probe and Mycobacterium tuberculosis complex- specific probe. There were 79.3 % （23/29） of nontuberculous mycobacteria which could be identified to the species level directly in the specimens from China. Some intraspecies heterogeneity in M. avium, M. intracellulare, M. chelonae and M. abscessus was observed. With the increase of sequences of internal transcribed spacer and numbers of whole microbial genomes, and further optimization of probes, the multiplex probe army will become a promising tool for the rapid and accurate identification of mycobacteria in ordinary clinical laboratories.展开更多
文摘Bovine tuberculosis (bTB) is an endemic zoonosis significantly affects animal health in Burkina Faso. The primary causative agent is Mycobacterium tuberculosis (M. tuberculosis) complex, mainly M. bovis. Cattle are considered as natural reservoir of M. bovis. However, in Burkina Faso, the circulation of these strains remains poorly understood and documented. This study aimed to identify and characterize Mycobacterium strains from suspected carcasses during routine meat inspection at Bobo-Dioulasso refrigerated slaughterhouse. A prospective cross-sectional study was conducted from January 2021 to December 2022 on cases of seizures linked to suspected bovine tuberculosis. Microbiological and molecular analyzes were used for mycobacterial strain isolation and characterization. Out of 50 samples, 24% tested positive by microscopy and 12% by culture. Molecular analysis identified 6 strains of Mycobacteria, exclusively Mycobacterium bovis specifically the subspecies bovis (Mycobacterium bovis subsp bovis). In conclusion, M. bovis subsp bovis is the primary agent responsible for bovine tuberculosis in Bobo-Dioulasso. Continuous monitoring of mycobacterial strains is therefore necessary for the effective control of this pathology in the local cattle population.
基金supported by the National Key Programme of Mega Infectious Diseases (2008ZX100/03-010-02)the National Natural Science Funding of China (30800029)
文摘Objective To identify the novel species ‘Mycobacterium fukienense＇ sp. nov of Mycobacterium chelonoe/abscessus complex from tuberculosis patients in Fujian Province, China. Methods Five of 27 clinical Mycobucterium isolates （CIs） were previously identified as M. chelonoe/obscessus complex by sequencing the hsp65, rpoB, 165-235 rRNA internal transcribed spacer region （its）, recA and sodA house-keeping genes commonly used to describe the molecular characteristics of Mycobocterium. Clinical Mycobecterium isolates were classified according to the gene sequence using a clustering analysis program. Sequence similarity within clusters and diversity between clusters were analyzed. Results The 5 isolates were identified with distinct sequences exhibiting 99.8% homology in the hsp65 gene. However, a complete lack of homology was observed among the sequences of the rpoB, 165-235 rRNA internal tronscribed spacer region （its）, sodA, and recA genes as compared with the M. obscessus. Furthermore, no match for rpoB, sodA, and recA genes was identified among the published sequences. Conclusion The novel species, Mycobacterium fukienense, is identified from tuberculosis patients in Fujian Province, China, which does not belong to any existing subspecies of M. cheloneo/abscessus complex.
基金Supported by Research Program in higher educational institutes of The Education Department of Hainan Province(No.Hj2010-21)Research Program of The Health Department of Hainan Province(No.2007-44)+1 种基金Natural Science Fund of Hainan Province(No.2008-30837)Cultivation Program of Hainan Medical University (HY2010-006)
文摘Objective:To clone and express Rv3265c gene of Mycobacterium tuberculosis in Escherichia coli (E.coli) under optimistic conditions,obtain and identify protein expressed,analyze the structure and characteristics of the protein using bioinformatics methods for future applications.Methods: Rv3265c gene from Mycobacterium tuberculosis H37Rv was amplified by polymerase chain reaction,and was cloned into the pET-30a vector after purification and recovery.The recombinant plasmid was sequenced and expressed in E.coli BL21(DE3),and then purified and identified by western blotting.The essential physical-chemical properties of the protein were predicated by bioinformatics tools,including subcellular location,secondary structure,domains,antigenic epitopes,etc.Tertiary structure of the protein based on homology modeling was estabUshed,while multi-sequence homological alignment and phylogenetic analysis were preformed.Results:The recombinant protein was obtained in soluble fraction from expression system in E.coli B121(DE3) carrying pET30- Rv3265c plasmid,and Rv3265c gene was expressed correctly.Bioinformatics analysis showed the protein contained no signal peptide and transmembrane helices,located outside of membrane.Secondary structure analysis revealed it containedα-helix,extended strand and random coil,46.8%,14.6%,38.6%,respectively.Furthermore,it possessed six potential antigenic epitopes,one glycosyl transferase domain.A simple three-dimensional model of this protein was constructed by Swiss-model sever.Both sequences and structures were conservative and especial either in gene or in protein.Conclusions:Rv3265c gene might be a desirable molecular target for anti-tuberculosis drug and vaccine.The purified protein from expression will be utilized to study the kinetics of L-rhamnosyltransferase and to develope an enzyme assay for screening vaccine or drug.
基金Supported by Research Program of The Health Department of Hainan Province(No.2007-44)Research Cultivation Program of Hainan Medical University(HY2010-006)+1 种基金Research Program in higher educational institutes of The Education Department of Hainan Province(No.Hj2010-21)Natural Science Fund of Hainan Province(No.2008~30837)
文摘Objective:To obtain fbpB-esxA fusing gene of Mycobacterium tuberculosis（MTU）,express the encoded fusing protein in Escherichia coli（E.coli）,identify protein acquired,and predict the structure and function of the protein utilizing methods of bioinformatics.Methods:fbpB and esxA gene were amplified from genome of MTB H37Rv by PCR.The fbpB-esxA fusing gene Iigated by（Gly<sub>4</sub>Ser）<sub>3</sub> linker was gained by means of Gene Splicing by Overlapping Extension PCU（SOEPCR）, and fusing gene was cloned into expression vector pET-30a.The recombinant plasmid was sequenced and expressed in E.coli BL21（DE3）.The protein was identified by Western blot using anti-HIS antibody.Secondary structure and antigenic epitopes of the protein were predicting using tools of bioinformatics.Results:The UNA sequences fbpB-esxA were identical with that published by GenBank.The Ag85B-ESAT-6 fusion protein about 50 kDa comprised 485 amino acids was efficiently produced from expression system in E.coli B1.21（DE3） under the induction of IPTG.Bioinformatics analysis showed the protein contained one transmembrane region and fourteen potential antigenic epitopes.Conclusions:The Ag85B-ESAT-6 fusion protein is successfully expressed with N-terminal HIS-tag.Gel filtration demonstrated that it exists as insoluble inclusion bodies mainly.The existence of linker doesn’t affect immunogenicity of Ag85B and ESAT-6.It will allow lor characterization in vitro and establish a foundation of further function research such as vaccine or diagnostic reagent.
基金supported by National Science and Technology Major Project of China[2018ZX10731301-002]。
文摘Objectives To evaluate the immunogenicity of Mycobacterium intracellulare proteins and determine the cross-reactive proteins between M.intracellulare and M.tuberculosis.Methods Protein extracts from M.intracellulare were used to immunize BALB/c mice.The antigens were evaluated using cellular and humoral immunoassays.The common genes between M.intracellular and M.tuberculosis were identified using genome-wide comparative analysis,and cross-reactive proteins were screened using immunoproteome microarrays.Results Immunization with M.intracellulare proteins induced significantly higher levels of the cytokines interferon-γ(IFN-γ),interleukin-2(IL-2),interleukin-12(IL-12),interleukin-6(IL-6)and immunoglobulins IgG,IgG1,IgM,and IgG2a in mouse serum.Bone marrow-derived macrophages isolated from mice immunized with M.intracellulare antigens displayed significantly lower bacillary loads than those isolated from mice immunized with adjuvants.Whole-genome sequence analysis revealed 396 common genes between M.intracellulare and M.tuberculosis.Microchip hybridization with M.tuberculosis proteins revealed the presence of 478 proteins in the serum of mice immunized with M.intracellulare protein extracts.Sixty common antigens were found using both microchip and genomic comparative analyses.Conclusion This is the advanced study to investigate the immunogenicity of M.intracellulare proteins and the cross-reactive proteins between M.intracellulare and M.tuberculosis.The results revealed the presence of a number of cross-reactive proteins between M.intracellulare and M.tuberculosis.Therefore,this study provides a new way of identifying immunogenic proteins for use in tuberculosis vaccines against both M.intracellulare and M.tuberculosis in future.
文摘Rapid differential identification of Mycobacterium species is essential for effective diagnosis and management of mycobacteriosis. The aim of this study was to develop a novel multiplex probe array based on the 16S-23S rRNA gene internal transcribed spacer sequence for the genotyping of mycobacteria to the species level. A pair of primers and a set of genus- and species-specific probes were designed from the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer, and 23S rRNA gene sequences of mycobacteria. We used a novel multiplex probe array for identification of 266 clinical specimens obtained from patients with mycobaterial infection. The results showed that the overall specificity and sensitivity of our novel probe array were both 100% for the genus-specific probe and Mycobacterium tuberculosis complex- specific probe. There were 79.3 % （23/29） of nontuberculous mycobacteria which could be identified to the species level directly in the specimens from China. Some intraspecies heterogeneity in M. avium, M. intracellulare, M. chelonae and M. abscessus was observed. With the increase of sequences of internal transcribed spacer and numbers of whole microbial genomes, and further optimization of probes, the multiplex probe army will become a promising tool for the rapid and accurate identification of mycobacteria in ordinary clinical laboratories.
