Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermo...Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.展开更多
Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture. Using a recently developed gene deletion strategy, two genes that encode the Glyco hydro_19 domain （GH19 domain） containing prot...Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture. Using a recently developed gene deletion strategy, two genes that encode the Glyco hydro_19 domain （GH19 domain） containing proteins, ghd-1 and ghd-2, were deleted separately and together from the F. columnare G4 wild type strain. Surprisingly, the single-, Aghd-1 and Aghd-2, and double-gene mutants, Aghd-1 Aghd-2, all had rhizoid and non-rhizoid colony morphotypes, which we named Aghd-1, Aghd-2, Aghd-1 Aghd-2, and NAghd-1, NAghd-2, and NAghd-1 Aghd-2. However, chitin utilization was not detected in either these mutants or in the wild type. Instead, skimmed milk degradation was observed for the mutants and the wild type; the non-rhizoid strain NAghd-2 exhibited higher degradation activity as revealed by the larger transparent circle on the skimmed milk plate. Using zebrafish as the model organism, we found that non-rhizoid mutants had higher LDs0 values and were less virulent because zebrafish infected with these survived longer. Transcriptome analysis between the non-rhizoid and rhizoid colony morphotypes of each mutant, i.e., NAghd-1 versus （vs） Aghd-1, NAghd-2 vs Aghd-2, and NAghd-1 Aghd-2 vs Aghd-1 Aghd-2, revealed a large number of differentially expressed genes, among which 39 genes were common in three of the pairs compared. Although most of these genes encode hypothetical proteins, a few molecules such as phage tail protein, rhs element Vgr protein, thiol-activated cytolysin, and TonB-dependent outer membrane receptor precursor, expression of which was down-regulated in non-rhizoid mutants but up-regulated in rhizoid mutants, may play a role F. columnare virulence.展开更多
Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable marke...Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.展开更多
Objective： The aim of the research was to investigate efficacy of dried bio-floc supplemented with immuno-stimulants as beta-glucan and nucleotide on mortality rate and relative percent survival （RPS） of tilapia in...Objective： The aim of the research was to investigate efficacy of dried bio-floc supplemented with immuno-stimulants as beta-glucan and nucleotide on mortality rate and relative percent survival （RPS） of tilapia infected with Flavobacterium columnare, VETSV01. Materials and Methods： A research was performed in 1-inch long tilapia stimulated its immunity by infection with Flavobacterium columnare, VETSV01. Pathogenesis of 0.2 mL Flavobacterium columnare, VETSV01 in the range of 10^2-10^8 mL-1 fish^-1 was injected into its intraperitoneal injection and intramuscular injection and suitable concentrations were screened to obtain for further experiment in a late restage. Based on data of minimal concentration, 50% mortality rate was caused and it was found the concentration of 108 mL^-1 fish^-1 caused 100% mortality rate and no lethal rate was found in other concentrations. Afterwards, efficacy of bio-floc sediment on survival rate of Tilapia infected with Flavobacterium columnare, VETSV01 was examined. Completely randomized design was employed and experiments were divided into 4 treatment： （1） contxol-commercial feed only, （2） commercial feed attached with dried bio-floc, （3） commercial feed attached with bio-floc and beta-glucan and （4） commercial feed attached with bio-floc and nucleotide. Experiment design was performed as 3 replicates with 10 fishes/replicates in 24 inch glass aquarium. Analysis of difference was performed and determination of mean differences of mortality rate mad relative percent survival was done through Duncan＇s Multiple Range Test （p 〈 0.05）. Results and Discussions： It was demonstrated feeding tilapia with commercial feed in combination with dried bio-floc with or without supplementation of 2 immuno-stimulants could lead to 100% survival rate and 100% relative percent survival rate and it was statistically significant different （p 〈 0.05） compared to control group. Bio-floc product supplemented with beta glucan or nucleotide could effectively stimulate immunity against Flavobacterium columnare, VETSV01 and such high effective performance is viewed as a promising potential product to be further developed and practically employed for sake of aquaculture industry in the near future.展开更多
Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide.Many studies have focused on the identification of protective antigens to aid in the development of nov...Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide.Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease.In this study,an immunoblotting approach was employed to identify immunogenic outer membrane proteins(OMPs)from F.columnare in two-dimensional electrophoresis(2-DE)map gels using antibacterial sera obtained from grass carp(Ctenopharyngodon idella),and anti-grass carp-recombinant Ig(rIg)monoclonal antibodies.Five unique immunogenic proteins,including the gliding motility lipoprotein GldJ(GldJ),hypothetical protein FCOL_13420(Fco1),lipoprotein(Lip),F0F1 ATP synthase subunit beta(F0f1)and outer membrane efflux protein precursor(Omep),were characterized.Over-expression of these proteins in Escherichia coli DE3,and their immunogenicity and protective efficacy were evaluated in grass carp.The relative percent survival(RPS)of the groups immunized separately with recombinant GldJ,Lip and Omep was 72%,64%and 68%,respectively when compared to control fish.Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F.columnare.The results suggest that GldJ,Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.展开更多
The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish, Siniperca chuatsi, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3...The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish, Siniperca chuatsi, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ε contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3γ/δ and CD3ε showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ. Additionally, while an N-glycosylation site was present in CD3c, the site was not observed in CD3γ/δ. The CD3γ/δ and CD3ε subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3γ/δ and CD3ε in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3γ/δ and CD3ε were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3γ/δ and CD3ε were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ε mR_NA, indicating that CD3γ/δ and CD3ε lymphocytes are involved in the immune response of this species.展开更多
基金Supported by the National Basic Research Program of China(973 Program)(No.2009CB118703)the Science and Technology Program of Xiamen Southern Oceanographic Center(No.14PYY050SF03)the National Science and Technology Support Program Project of China(No.2012BAD25B02)
文摘Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.
基金Supported by the Chinese Academy of Sciences(No.XDA08010207)the National Key Technology R&D Program of China(No.2012BAD25B02)the State Key Laboratory of Freshwater Ecology and Biotechnology(No.2016FBZ04)
文摘Flavobacterium columnare is the pathogenic agent of columnaris disease in aquaculture. Using a recently developed gene deletion strategy, two genes that encode the Glyco hydro_19 domain （GH19 domain） containing proteins, ghd-1 and ghd-2, were deleted separately and together from the F. columnare G4 wild type strain. Surprisingly, the single-, Aghd-1 and Aghd-2, and double-gene mutants, Aghd-1 Aghd-2, all had rhizoid and non-rhizoid colony morphotypes, which we named Aghd-1, Aghd-2, Aghd-1 Aghd-2, and NAghd-1, NAghd-2, and NAghd-1 Aghd-2. However, chitin utilization was not detected in either these mutants or in the wild type. Instead, skimmed milk degradation was observed for the mutants and the wild type; the non-rhizoid strain NAghd-2 exhibited higher degradation activity as revealed by the larger transparent circle on the skimmed milk plate. Using zebrafish as the model organism, we found that non-rhizoid mutants had higher LDs0 values and were less virulent because zebrafish infected with these survived longer. Transcriptome analysis between the non-rhizoid and rhizoid colony morphotypes of each mutant, i.e., NAghd-1 versus （vs） Aghd-1, NAghd-2 vs Aghd-2, and NAghd-1 Aghd-2 vs Aghd-1 Aghd-2, revealed a large number of differentially expressed genes, among which 39 genes were common in three of the pairs compared. Although most of these genes encode hypothetical proteins, a few molecules such as phage tail protein, rhs element Vgr protein, thiol-activated cytolysin, and TonB-dependent outer membrane receptor precursor, expression of which was down-regulated in non-rhizoid mutants but up-regulated in rhizoid mutants, may play a role F. columnare virulence.
基金Supported by the National Basic Research Program of China(973Program)(No.2009CB118703)
文摘Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.
文摘Objective： The aim of the research was to investigate efficacy of dried bio-floc supplemented with immuno-stimulants as beta-glucan and nucleotide on mortality rate and relative percent survival （RPS） of tilapia infected with Flavobacterium columnare, VETSV01. Materials and Methods： A research was performed in 1-inch long tilapia stimulated its immunity by infection with Flavobacterium columnare, VETSV01. Pathogenesis of 0.2 mL Flavobacterium columnare, VETSV01 in the range of 10^2-10^8 mL-1 fish^-1 was injected into its intraperitoneal injection and intramuscular injection and suitable concentrations were screened to obtain for further experiment in a late restage. Based on data of minimal concentration, 50% mortality rate was caused and it was found the concentration of 108 mL^-1 fish^-1 caused 100% mortality rate and no lethal rate was found in other concentrations. Afterwards, efficacy of bio-floc sediment on survival rate of Tilapia infected with Flavobacterium columnare, VETSV01 was examined. Completely randomized design was employed and experiments were divided into 4 treatment： （1） contxol-commercial feed only, （2） commercial feed attached with dried bio-floc, （3） commercial feed attached with bio-floc and beta-glucan and （4） commercial feed attached with bio-floc and nucleotide. Experiment design was performed as 3 replicates with 10 fishes/replicates in 24 inch glass aquarium. Analysis of difference was performed and determination of mean differences of mortality rate mad relative percent survival was done through Duncan＇s Multiple Range Test （p 〈 0.05）. Results and Discussions： It was demonstrated feeding tilapia with commercial feed in combination with dried bio-floc with or without supplementation of 2 immuno-stimulants could lead to 100% survival rate and 100% relative percent survival rate and it was statistically significant different （p 〈 0.05） compared to control group. Bio-floc product supplemented with beta glucan or nucleotide could effectively stimulate immunity against Flavobacterium columnare, VETSV01 and such high effective performance is viewed as a promising potential product to be further developed and practically employed for sake of aquaculture industry in the near future.
文摘Flavobacterium columnare is a Gram-negative bacterium that causes columnaris disease in freshwater fish worldwide.Many studies have focused on the identification of protective antigens to aid in the development of novel vaccines against the disease.In this study,an immunoblotting approach was employed to identify immunogenic outer membrane proteins(OMPs)from F.columnare in two-dimensional electrophoresis(2-DE)map gels using antibacterial sera obtained from grass carp(Ctenopharyngodon idella),and anti-grass carp-recombinant Ig(rIg)monoclonal antibodies.Five unique immunogenic proteins,including the gliding motility lipoprotein GldJ(GldJ),hypothetical protein FCOL_13420(Fco1),lipoprotein(Lip),F0F1 ATP synthase subunit beta(F0f1)and outer membrane efflux protein precursor(Omep),were characterized.Over-expression of these proteins in Escherichia coli DE3,and their immunogenicity and protective efficacy were evaluated in grass carp.The relative percent survival(RPS)of the groups immunized separately with recombinant GldJ,Lip and Omep was 72%,64%and 68%,respectively when compared to control fish.Up-regulation of immuno-related genes and specific antibodies were detected in immunized fish and sera of immunized fish inhibited the growth of F.columnare.The results suggest that GldJ,Lip and Omep are major protective antigens and may be considered as novel candidates in the development of vaccines against columnaris disease in fish.
基金Supported by the National Natural Science Foundation of China (No.U0631010),the Government of Guangdong Provincethe National Basic Research Program of China (973 Program) (No. 2009CB118703)
文摘The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish, Siniperca chuatsi, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ε contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3γ/δ and CD3ε showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ. Additionally, while an N-glycosylation site was present in CD3c, the site was not observed in CD3γ/δ. The CD3γ/δ and CD3ε subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3γ/δ and CD3ε in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3γ/δ and CD3ε were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3γ/δ and CD3ε were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ε mR_NA, indicating that CD3γ/δ and CD3ε lymphocytes are involved in the immune response of this species.
