The caspase gene family is a crucial gene cluster that regulates apoptosis which contribute to programmed cell death,cell proliferation and differentiation,and several immune responses.In our study,a complete set of 1...The caspase gene family is a crucial gene cluster that regulates apoptosis which contribute to programmed cell death,cell proliferation and differentiation,and several immune responses.In our study,a complete set of 12 caspase genes were identified in spotted sea bass Lateolabrax maculatus.These genes were divided into three subfamilies:2 inflammatory caspases(casp-1 and casp-14-like),5 apoptosis initiators(casp-2,casp-8a,casp-8b,casp-9,and casp-10),and 5 apoptosis executioners(casp-3a,casp-3b,casp-3-like,casp-6,and casp-7).Their phylogenetic relationships,synteny and gene structures were systematically analyzed.Furthermore,the relative expression profiles of the caspase family members in the liver,intestine,head kidney,and spleen were measured by q PCR after infection with Vibrio harveyi.The results showed that the overall mRNA levels of the caspase genes were dramatically increased after V.harveyi infection,and the expression patterns varied among genes and tissues.More caspase genes underwent pronounced expression changes in the head kidney and spleen than in the liver or intestine,mainly after 48 h of the challenge.Specifically,casp-3a,casp-3b,casp-3-like,casp-6,casp-7,casp-8a,casp-8b,casp-10,and casp-14-like in the head kidney,and casp-3-like,casp-6,casp-7,and casp-14-like in the spleen,were the most responsive caspase genes which may contribute significantly to immune regulation in spotted sea bass.Additionally,the apoptosis level in head kidney and spleen after infection were examined using the Caspase assay.Our study provides a systemic overview of the caspase gene family in spotted sea bass after V.harveyi infection and lays a foundation for further deciphering the biological roles of these caspase genes.展开更多
Vibrio harveyi, known as a pathogenic bacterium caused severe secondary bacterial infections of the large yellow croaker Larimichthys crocea, was identified as an endosymbiont in the marine parasitic ciliate protozoan...Vibrio harveyi, known as a pathogenic bacterium caused severe secondary bacterial infections of the large yellow croaker Larimichthys crocea, was identified as an endosymbiont in the marine parasitic ciliate protozoan Cryptocaryon irritans. Meta 16 S sequencing method was used to identify the bacterial flora in C. irritans, and V.harveyi was isolated via culture-dependent method. Vibrio harveyi was observed in cytoplasm of C. irritans at the stage of tomont both by transmission electron microscopy and by Fluorescence in situ hybridization; no signal,however, was detected in nucleus area. The relationship between V. harveyi and C. irritans and the role of endosymbiotic V. harveyi in C. irritans merit further investigation.展开更多
Vibrio harveyi is a pathogen of various aquatic organisms that has been recently associated with massive mortality episodes in the aquaculture industry.Recurrent outbreaks of vibriosis are closely correlated with the ...Vibrio harveyi is a pathogen of various aquatic organisms that has been recently associated with massive mortality episodes in the aquaculture industry.Recurrent outbreaks of vibriosis are closely correlated with the capacity of this bacterial species to survive long-term starvation conditions.To study the regulation mechanism of gene expression at the transcriptional level in V.harveyi under starvation conditions,the transcriptomic response profiles were determined of the Portunus trituberculatus pathogen V.harveyi strain DY1 under normal conditions and after four weeks of starvation.A total of 4679 and 4661 genes were expressed in the non-starved and starved cells,respectively.The significantly differentially expressed genes(DEGs)between non-starved and starved groups were identified,in which 255 genes were up-regulated and 411 genes were down-regulated.GO analysis and KEGG enrichment analysis were used to analyze the DEGs and revealed the involvement of these DEGs in many pathways,including ABC transporters,flagellum assembly,and fatty acid metabolism.Several DEGs were randomly selected and their expression levels were confirmed by quantitative real-time PCR(qRT-PCR).This is the first comprehensive transcriptomic analysis of starvation ef fects in V.harveyi.Our findings will facilitate future study on stress adaptation and survival mechanisms of V.harveyi.展开更多
An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phosph...An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from the genome of V.harveyi SF-1.The gene consisted of 1665 base pairs and encoded a 554 amino acid polypeptide,which showed a high similarity to those of the OppAs of V.harveyi and other Vibrio species.The gene was subcloned into pET-28a(+)and expressed in Escherichia coli.The expressed recombinant protein was purified by Ni-NTA metal affinity chro-matography.The expressed recombinant protein showed a 58 kDa band on SDS-PAGE and exhibited phospholipase C activity with the optima of temperature 50℃ and pH 8.0.The purified protein was toxic to the flounder gill cells.An OppA mutant of V.harveyi SF-1 was constructed by homologous recombination.The mutant strain was less virulent when it was intraperitoneally inoculated to zebra fish,with the LD50 of 5.46×105 CFU fish-1,compared to 3.11×104 CFU fish-1 of the wild-type strain,which indicated that the OppA might play an important role in the pathogenicity of V.harveyi.展开更多
Hepcidins are small cysteine-rich antimicrobial peptides that play a vital role in immunity against pathogen invasion.Here,a hepcidin(Cshep)from Centropristis striata was described,which is considered as a valuable aq...Hepcidins are small cysteine-rich antimicrobial peptides that play a vital role in immunity against pathogen invasion.Here,a hepcidin(Cshep)from Centropristis striata was described,which is considered as a valuable aquaculture marine species in China.The open reading frame consisted of 273 bp.Eight conserved cysteine residues were identified.Phylogenetic analysis showed that Cshep had a relatively close relationship with the hepcidin from Epinephelus moara.Quantitative real-time PCR analysis demonstrated that Cshep was highly expressed in liver and significantly up-regulated when challenged with Vibrio harveyi.In addition,the synthetic Cshep peptide had a high antimicrobial activity against V.harveyi,but low against other pathogenic bacteria tested in this study.The killing kinetics analysis revealed that Cshep had a fast bactericidal effect on V.harveyi.These results suggested that Cshep may be involved in the immune response of C.striata against V.harveyi infection.展开更多
[Objective] This study aimed to analyze the in vitro inhibitory activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and its biofilms. [Result] By agar diffusion test,in vitro inhibitory ac...[Objective] This study aimed to analyze the in vitro inhibitory activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and its biofilms. [Result] By agar diffusion test,in vitro inhibitory activity of S.chinensis and P. sibiricum against V. harveyi was investigated. The minimal inhibitory concentration(MIC) a nd minimal bactericidal concentration(MBC) of S.chinensis and P.sibiricum against V.harveyi were determined by doubling dilution method. The inhibitory activity of S. chinensis and P.sibiricum on the formation of V.harveyi biofilms was evaluated by modified MTT assay. [Result]Both S.chinensis and P.sibiricum had inhibitory activity against V.harveyi. The inhibition zone diameter of S.chinensis against V.harveyi was 17.95 mm; MIC and MBC of S.chinensis were both 3.125 mg / ml. The inhibition zone diameter of P. sibiricum against V.harveyi was 12.22 mm; MIC and MBC of P.sibiricum were 3.125 and 6. 250 mg / ml,respectively. When the concentration was higher than 6. 25 mg / ml,S.chinensis decoction had extremely significant inhibitory activity against V.harveyi( P < 0. 01); when the concentration was higher than 3. 125 mg / ml,P. sibiricum had extremely significant inhibitory activity against V. harveyi(P <0. 01). [Conclusion]S.chinensis and P.sibiricum could significantly inhibit V.harveyi and its biofilms.展开更多
Chemotactic motility is involved in the virulence of many pathogenic bacteria. In order to understand the role of chemotactic motility of Vibrio harveyi in cellular processes and virulence, mini-Tn10/Kan transposon-in...Chemotactic motility is involved in the virulence of many pathogenic bacteria. In order to understand the role of chemotactic motility of Vibrio harveyi in cellular processes and virulence, mini-Tn10/Kan transposon-induced mutants with deficient chemotactic motility were constructed, screened, and identified. Sequence analysis revealed that the 465-bp fragment (GenBank accession number HM630274) flanking the transposon insertion site in mutant TS-CM1 had the highest identity (96.9%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (91.8%) with the pgk gene of V. parahaemolyticus RIMD 2210633. In another mutant, TS-CM2, 356 bp of transposon-flanking sequence (GenBank accession number HM630275) also showed the highest identity (94.6%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (92.4%) with the flaB gene of V. alginolyticus HY9901. Studies on virulence-related biological characteristics such as growth, motility, adhesion, and infectivity of the mutants showed that disruption of either the flagellin gene or energy metabolism gene led to subsequent loss of chemotactic motility and changes in growth, motility, adhesion, and virulence of the pathogenic V. harveyi. Hence, the flagellin gene and crucial energy metabolism gene played an important role in the chemotactic motility of V. harveyi.展开更多
The FlaA gene from Vibrio harveyi,with a short nucleotide sequence encoding the Flag marker,was cloned into the eukaryotic expression vector pcDNA3.1(+) (designated as pcFlaA).Ninety grouper (Epinephelus awoara) were ...The FlaA gene from Vibrio harveyi,with a short nucleotide sequence encoding the Flag marker,was cloned into the eukaryotic expression vector pcDNA3.1(+) (designated as pcFlaA).Ninety grouper (Epinephelus awoara) were separated into three equal size groups.An experimental group was immunized with pcFlaA,Control I group was immunized with the vector pcDNA3.1(+),and Control II group was immunized with PBS.The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry.We also evaluated the immunogenicity and protective efficacy of pcFlaA against V.harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test.We successfully transfected the fish muscle with pcFlaA.The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection.The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II.Furthermore,the immunized fish generated specific antibody.The vaccination also resulted in significantly higher survival during the bacterial challenge test.展开更多
[Objective] This study was conducted to determine the in-vitro inhibitory effects of Prunus mume,Coptis chinensis and Crataegus pinnatifida on Vibrio harveyi and its biofilm. [Method]The inhibitory zone diameters of t...[Objective] This study was conducted to determine the in-vitro inhibitory effects of Prunus mume,Coptis chinensis and Crataegus pinnatifida on Vibrio harveyi and its biofilm. [Method]The inhibitory zone diameters of the three Chinese herbal medicines against V. harveyi were determined by agar diffusion method; the minimal inhibitory concentration( MIC) and minimal bactericidal concentration( MBC) values of the three Chinese herbal medicines against V. harveyi were determined by doubling dilution method; and the effects of the three Chinese herbal medicines on the formation of V. harveyi biofilm were determined by methyl thiazolyl tetrazolium( MTT) method. [Result]The three Chinese herbal medicines all inhibited V. harveyi to different degrees. C. chinensis and C. pinnatifida and P. mume exhibited the inhibitory zone diameters of( 17. 62 ± 0. 04),( 20. 16 ± 0. 08) and( 30. 76 ± 0. 26) mm against V. harveyi,respectively. P. mume and C. pinnatifida had strong inhibitory effects on V. harveyi. The MIC and MBC values of P. mume against V. harveyi were 7. 812 5 mg/ml; the MIC and MBC values of C. pinnatifida against V. harveyi were 31. 25 mg/ml; and the MIC and MBC values of C. chinensis against V. harveyi were 62. 5 mg/ml. P. mume had the strongest antibacterial and bactericidal ability. The MIC values of C. pinnatifida,C. chinensis and P. mume against V. harveyi were 7. 81,7. 81 and 1. 96 mg/ml,respectively,i. e.,P. mume exhibited the lowest MIC. [Conclusion] P. mume,C. pinnatifida and C. chinensis all have inhibitory effects on V. harveyi and its biofilm,and P. mume has the strongest bactericidal ability.展开更多
Vibrio harveyi cells (dose>103 cells mL -1) and extracellular products (ECP; >25 μg mL -1 of total protein concentration) destroyed haematopoietic cultures of Nephrops norvegicus within 24 h of exposure. Cytopa...Vibrio harveyi cells (dose>103 cells mL -1) and extracellular products (ECP; >25 μg mL -1 of total protein concentration) destroyed haematopoietic cultures of Nephrops norvegicus within 24 h of exposure. Cytopathic effects (CPE) started after 4 h of exposure to the bacterial cells, with some granularity in the cytoplasm, mostly in cells in the outer periphe-ry of the explant growth. At the end of the infection, a considerable number of nuclei remained attached to the substrate, apparently unaffected. Following exposure to ECP, initial deterioration was observed at 2 h with the presence of granularity in the cytoplasm of<20% cells, and few cells displayed small vacuoles around the nuclei. Parallel results were obtained using whole animal experiments, with V. harveyi cells being lethal to nephrops within 24 h.展开更多
Several reports have revealed the vital role that probiotics play in fish growth and health.However,few works are available for host gut-derived probiotics on the growth,immunity,and gut microbiota of fish,especially ...Several reports have revealed the vital role that probiotics play in fish growth and health.However,few works are available for host gut-derived probiotics on the growth,immunity,and gut microbiota of fish,especially in hybrid grouper (♀Epinephelus fuscoguttatus×♂Epinephelus lanceolatus) due to their isolation difficulty and functional verification.This study aimed at assessing 3 host gut-derived Bacillus species?effects on the growth,immune and antioxidant-biochemical responses,haematological parameters,intestinal morphology,immune-related gene expression,gut microbiota,and disease resistance against Vibrio harveyi in hybrid grouper.A total of 480 hybrid grouper (initial weight=9.03±0.02 g) were randomly allotted into 4 groups,namely,the group fed a basal diet without probiotic inclusion (control,B0),the group fed the basal diet with Bacillus velezensis GPSAK4 (BV),the group fed the basal diet with Bacillus subtilis GPSAK9 (BS),and the group fed the basal diet with Bacillus tequilensis GPSAK2 (BT) strains at 1.0×10^(9)CFU/g.After a 6-week feeding trial,the results revealed significant improvements (P<0.05) in the growth performance,whole fish-body proximate composition,blood haematological parameters,serum,liver,and intestinal biochemical indexes,intestinal morphology,and protection against V.harveyi pathogen in the probiotic-treated groups compared with the untreated.Additionally,the expressions of intestinal tight junction genes (occludin and ZO1),pro-and anti-inflammatory genes,including IL1β,IL6,IL8,TNFa,MyD88,IL10,and TGFβ,were upregulated (P<0.05) after Bacillus species administration.Host gut-derived Bacillus supplementation shaped the gut microbiota by significantly increasing (P<0.05) the relative abundance of Proteobacteria,Bacteroidetes,Actinobacteria (except the BS group),Acidobacteria(except the BT group),Cyanobacteria (except the BV and BT groups),and Verrucomicrobia phyla,as well as known beneficial genera (Romboutsia,Turicibacter,Epulopiscium,Clostridium_sensu_stricto 1 and 13,Lactobacillus,and Bacillus),but significantly decreased (P<0.05) the abundance of Firmicutes,Chloroflexi,and Fusobacteria phyla,and purported pathogenic genera (Staphylococcus and Photobacterium) compared with the control group.Collectively,the results suggest that B.velezensis GPSAK4,B.subtilis GPSAK9(especially this strain),B.tequilensis GPSAK2 dietary supplementation at 1.0×10^(9)CFU/g has positive effects on the intestinal health of hybrid grouper via microbial composition modulation,thus enhancing the assimilation and absorption of nutrients to boost fish growth,immunity,and disease resistance.展开更多
Turbot Scophthalmus maximus is an important mariculture fish species with high economic value.However,the bacterial diseases caused by Vibrio anguillarum infection bring huge economic losses to the turbot aquaculture ...Turbot Scophthalmus maximus is an important mariculture fish species with high economic value.However,the bacterial diseases caused by Vibrio anguillarum infection bring huge economic losses to the turbot aquaculture industry.To understand the immune response of the turbot against V.anguillarum infection and to explore novel immune-related genes,the transcriptome analysis of turbot spleen and gills were conducted after V.anguillarum infection.Differentially expressed genes(DEGs)were identified in spleen and gill of the turbot amounted to 17261 and 16436,respectively.A large number of immunerelated DEGs were enriched in cytokine-cytokine receptor interaction signaling pathway,and the others by the kyoto encyclopedia of genes and genomes(KEGG)enrichment.The gene ontology(GO)classification analysis revealed that V.anguillarum infection had the greatest effect on biological processes and cellular components.Twelve immune-related DEGs were identified in the spleen(cstl.1,egfl6,lamb21,v2rx4,calcr,and gpr78a)and gills(ghra,sh3gl2a,cst12,inhbaa,cxcl8,and il-1b)by heat map.The proteinprotein interaction(PPI)networks were constructed to analyze the immune mechanism.The results demonstrate that the maturation and antigen processing of major histocompatibility complex(MHC)class II molecule,and calcitonin-or adrenomedullin-regulated physiological activity were important events in the immunity of turbot against V.anguillarum infection.In the gills,the protein interactions in TGF-βsignaling pathway,production of inflammatory factors,and endocytosis regulation were most significant.Our research laid a foundation for discovering novel immune-related genes and enriching the knowledge of immune mechanisms of turbot against V.anguillarum infection.展开更多
Vibrio harveyi,the major causative agent of vib-riosis,affects a diverse range of marine cultured organisms over a wide geographical area.However,reports about screening the effective antigen and research on vaccines ...Vibrio harveyi,the major causative agent of vib-riosis,affects a diverse range of marine cultured organisms over a wide geographical area.However,reports about screening the effective antigen and research on vaccines of V.harveyi are scarce.Flagellin,lipopolysaccharide(LPS)and outer membrane proteins(OMP)are major immunogenic antigens in many Gram-negative bacteria.In this study,the flagellin,OMP and LPS of the V.harveyi TS-628 strain iso-lated from infected groupers were extracted and Western blot analysis was used to detect the antigenicity of these extrac-tions.Results of the Western blot assay reveal that there are four positive flagellin bands:35 kDa,38 kDa,43 kDa,and 52 kDa,of which the 43 kDa and 52 kDa bands displayed the strongest positive reaction.There are five positive OMPbands about 35 kDa,38 kDa,43 kDa,47 kDa,and 52 kDa,of which the 43 kDa appeared to have the strongest positive reaction although the other four proteins also displayed strong reac-tions.However,LPS is Western blot-negative.These results indicate that the 43 kDa and 52 kDa flagellin and OMP of size 43 kDa,52 kDa can be candidates for developing vaccines against V.harveyi.展开更多
Objective:To evaluate the in vitro biofilm forming capacity on abiotic food contact surfaces by Vibrio harveyi(V.harveyi)strains.Methods:Thirty six Gram-negative V.harveyi strains were isolated from various street ven...Objective:To evaluate the in vitro biofilm forming capacity on abiotic food contact surfaces by Vibrio harveyi(V.harveyi)strains.Methods:Thirty six Gram-negative V.harveyi strains were isolated from various street vended seafood outlets in a food processing line and evaluated for their ability to produce mucoid biofilms on food contact surfaces using a microplate assay.Phenotypic characterization of mucoid biofilm producing V.harveyi strains were screened on Congo red agar,thiosulfate-citrate-bile salts-sucrose agar and tryptic soy agar,respectively.Results:Only five V.harveyi strains(14%)were mucoid biofilm producers characterized by formation of black colonies,whereas the remaining 31 strains(86%)were not capable of producing biofilm characterized by formation of red colonies or pinkish-red colonies with darkening at the centre.The morphological,physiological and biochemical characteristics of these isolates were studied using standard protocols.Strain identification was confirmed by polymerase chain reaction targeted to species-specific polymerase chain reaction primers VH-1 and VH-2 corresponding to variable regions of V.harveyi 16S rRNA sequence.All the biofilm-forming strains showed resistance to at least three antimicrobial compounds tested.V.harveyi strains isolated from various seafood were able to form biofilms of different capacity,and the strains VB267,VB238 and VB166 isolated from cat fish,shrimp and eel fish exhibited significantly greater biofilm forming ability compared to other isolates.Conclusions:It can be concluded from the present study that the strain VB166 was able to better attach and form subsequent biofilms on glass and stainless steel compared to high density polyethylene.These properties allow these bacteria to survive,proliferate and persist in street vended seafood outlets.展开更多
基金the National Key R&D Program of China(No.2020YFD0900204)the National Natural Science Foundation of China(No.32072947)+1 种基金the China Agriculture Research System of MOF and MARA(No.CARS-47)the KU-OUC Dual Master’s Program and Ocean University of China Scholarship Council。
文摘The caspase gene family is a crucial gene cluster that regulates apoptosis which contribute to programmed cell death,cell proliferation and differentiation,and several immune responses.In our study,a complete set of 12 caspase genes were identified in spotted sea bass Lateolabrax maculatus.These genes were divided into three subfamilies:2 inflammatory caspases(casp-1 and casp-14-like),5 apoptosis initiators(casp-2,casp-8a,casp-8b,casp-9,and casp-10),and 5 apoptosis executioners(casp-3a,casp-3b,casp-3-like,casp-6,and casp-7).Their phylogenetic relationships,synteny and gene structures were systematically analyzed.Furthermore,the relative expression profiles of the caspase family members in the liver,intestine,head kidney,and spleen were measured by q PCR after infection with Vibrio harveyi.The results showed that the overall mRNA levels of the caspase genes were dramatically increased after V.harveyi infection,and the expression patterns varied among genes and tissues.More caspase genes underwent pronounced expression changes in the head kidney and spleen than in the liver or intestine,mainly after 48 h of the challenge.Specifically,casp-3a,casp-3b,casp-3-like,casp-6,casp-7,casp-8a,casp-8b,casp-10,and casp-14-like in the head kidney,and casp-3-like,casp-6,casp-7,and casp-14-like in the spleen,were the most responsive caspase genes which may contribute significantly to immune regulation in spotted sea bass.Additionally,the apoptosis level in head kidney and spleen after infection were examined using the Caspase assay.Our study provides a systemic overview of the caspase gene family in spotted sea bass after V.harveyi infection and lays a foundation for further deciphering the biological roles of these caspase genes.
基金The National Natural Science Foundation of China under contract Nos 31372504,41176115 and 41476118
文摘Vibrio harveyi, known as a pathogenic bacterium caused severe secondary bacterial infections of the large yellow croaker Larimichthys crocea, was identified as an endosymbiont in the marine parasitic ciliate protozoan Cryptocaryon irritans. Meta 16 S sequencing method was used to identify the bacterial flora in C. irritans, and V.harveyi was isolated via culture-dependent method. Vibrio harveyi was observed in cytoplasm of C. irritans at the stage of tomont both by transmission electron microscopy and by Fluorescence in situ hybridization; no signal,however, was detected in nucleus area. The relationship between V. harveyi and C. irritans and the role of endosymbiotic V. harveyi in C. irritans merit further investigation.
基金Supported by the Jiangsu Agricultural Industry Technology System(No.02)the National Natural Science Foundation of China(No.31972830)+3 种基金the Natural Science Foundation of Jiangsu Province(No.BK20180915)the Jiangsu Agriculture Science and Technology Innovation Fund(No.CX(18)2012)the Fishery Science and Technology Innovation Projects of Jiangsu Province(No.D2017-3)the “Blue Project” of Yangzhou University and the Projects of Shuichan Sanxin of Jiangsu Province(No.Y2016-33,D2017-3)
文摘Vibrio harveyi is a pathogen of various aquatic organisms that has been recently associated with massive mortality episodes in the aquaculture industry.Recurrent outbreaks of vibriosis are closely correlated with the capacity of this bacterial species to survive long-term starvation conditions.To study the regulation mechanism of gene expression at the transcriptional level in V.harveyi under starvation conditions,the transcriptomic response profiles were determined of the Portunus trituberculatus pathogen V.harveyi strain DY1 under normal conditions and after four weeks of starvation.A total of 4679 and 4661 genes were expressed in the non-starved and starved cells,respectively.The significantly differentially expressed genes(DEGs)between non-starved and starved groups were identified,in which 255 genes were up-regulated and 411 genes were down-regulated.GO analysis and KEGG enrichment analysis were used to analyze the DEGs and revealed the involvement of these DEGs in many pathways,including ABC transporters,flagellum assembly,and fatty acid metabolism.Several DEGs were randomly selected and their expression levels were confirmed by quantitative real-time PCR(qRT-PCR).This is the first comprehensive transcriptomic analysis of starvation ef fects in V.harveyi.Our findings will facilitate future study on stress adaptation and survival mechanisms of V.harveyi.
基金supported by grants of the National High-Tech R and D Program (2007AA09Z416)the Natural Science Foundation of China (30972275)
文摘An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from the genome of V.harveyi SF-1.The gene consisted of 1665 base pairs and encoded a 554 amino acid polypeptide,which showed a high similarity to those of the OppAs of V.harveyi and other Vibrio species.The gene was subcloned into pET-28a(+)and expressed in Escherichia coli.The expressed recombinant protein was purified by Ni-NTA metal affinity chro-matography.The expressed recombinant protein showed a 58 kDa band on SDS-PAGE and exhibited phospholipase C activity with the optima of temperature 50℃ and pH 8.0.The purified protein was toxic to the flounder gill cells.An OppA mutant of V.harveyi SF-1 was constructed by homologous recombination.The mutant strain was less virulent when it was intraperitoneally inoculated to zebra fish,with the LD50 of 5.46×105 CFU fish-1,compared to 3.11×104 CFU fish-1 of the wild-type strain,which indicated that the OppA might play an important role in the pathogenicity of V.harveyi.
基金The Youth Foundation of Guangxi Zhuang Autonomous Region,China under contract No.2018GXNSFBA050032the Innovation Driven Development Foundation of Guangxi under contract Nos AD19245135,AD19245161+1 种基金the Doctoral Research Startup Fund of the Fourth Institute of Oceanography,Ministry of Natural Resources under contract Nos 201803,201806the Fund of Key Laboratory of South China Sea Fishery Resources Exploitation&Utilization,Ministry of Agriculture and Rural Affairs,China under contract No.FREU2016-04。
文摘Hepcidins are small cysteine-rich antimicrobial peptides that play a vital role in immunity against pathogen invasion.Here,a hepcidin(Cshep)from Centropristis striata was described,which is considered as a valuable aquaculture marine species in China.The open reading frame consisted of 273 bp.Eight conserved cysteine residues were identified.Phylogenetic analysis showed that Cshep had a relatively close relationship with the hepcidin from Epinephelus moara.Quantitative real-time PCR analysis demonstrated that Cshep was highly expressed in liver and significantly up-regulated when challenged with Vibrio harveyi.In addition,the synthetic Cshep peptide had a high antimicrobial activity against V.harveyi,but low against other pathogenic bacteria tested in this study.The killing kinetics analysis revealed that Cshep had a fast bactericidal effect on V.harveyi.These results suggested that Cshep may be involved in the immune response of C.striata against V.harveyi infection.
基金Supported by Project of Enhancing School with Innovation of Guangdong Ocean University(GDOU2015050216)Major Program for the Fundamental Research of the Department of Education of Guangdong Province(2014GKXM046)International Cooperation Innovation Platform Project of Universities in Guangdong Province(2013gjhz0008)
文摘[Objective] This study aimed to analyze the in vitro inhibitory activity of Shisandra chinensis and Polygonatum sibiricum against Vibrio harveyi and its biofilms. [Result] By agar diffusion test,in vitro inhibitory activity of S.chinensis and P. sibiricum against V. harveyi was investigated. The minimal inhibitory concentration(MIC) a nd minimal bactericidal concentration(MBC) of S.chinensis and P.sibiricum against V.harveyi were determined by doubling dilution method. The inhibitory activity of S. chinensis and P.sibiricum on the formation of V.harveyi biofilms was evaluated by modified MTT assay. [Result]Both S.chinensis and P.sibiricum had inhibitory activity against V.harveyi. The inhibition zone diameter of S.chinensis against V.harveyi was 17.95 mm; MIC and MBC of S.chinensis were both 3.125 mg / ml. The inhibition zone diameter of P. sibiricum against V.harveyi was 12.22 mm; MIC and MBC of P.sibiricum were 3.125 and 6. 250 mg / ml,respectively. When the concentration was higher than 6. 25 mg / ml,S.chinensis decoction had extremely significant inhibitory activity against V.harveyi( P < 0. 01); when the concentration was higher than 3. 125 mg / ml,P. sibiricum had extremely significant inhibitory activity against V. harveyi(P <0. 01). [Conclusion]S.chinensis and P.sibiricum could significantly inhibit V.harveyi and its biofilms.
基金The National Natural Science Foundation of China under contract Nos 31272699 and 41176115National Department Public Benefit Research Foundation of China under contract No. 200903029+1 种基金the Natural Science Foundation of Fujian Province under contract No.2011J06014the National Hi-Tech Research and Development Program of China (863 Program) under contract No. 2007AA09Z115
文摘Chemotactic motility is involved in the virulence of many pathogenic bacteria. In order to understand the role of chemotactic motility of Vibrio harveyi in cellular processes and virulence, mini-Tn10/Kan transposon-induced mutants with deficient chemotactic motility were constructed, screened, and identified. Sequence analysis revealed that the 465-bp fragment (GenBank accession number HM630274) flanking the transposon insertion site in mutant TS-CM1 had the highest identity (96.9%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (91.8%) with the pgk gene of V. parahaemolyticus RIMD 2210633. In another mutant, TS-CM2, 356 bp of transposon-flanking sequence (GenBank accession number HM630275) also showed the highest identity (94.6%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (92.4%) with the flaB gene of V. alginolyticus HY9901. Studies on virulence-related biological characteristics such as growth, motility, adhesion, and infectivity of the mutants showed that disruption of either the flagellin gene or energy metabolism gene led to subsequent loss of chemotactic motility and changes in growth, motility, adhesion, and virulence of the pathogenic V. harveyi. Hence, the flagellin gene and crucial energy metabolism gene played an important role in the chemotactic motility of V. harveyi.
基金Supported by Fujian Science and Technology Innovation Foundation for Young Scientists (No.2006F3096)Scientific Research Foundation of Jimei University
文摘The FlaA gene from Vibrio harveyi,with a short nucleotide sequence encoding the Flag marker,was cloned into the eukaryotic expression vector pcDNA3.1(+) (designated as pcFlaA).Ninety grouper (Epinephelus awoara) were separated into three equal size groups.An experimental group was immunized with pcFlaA,Control I group was immunized with the vector pcDNA3.1(+),and Control II group was immunized with PBS.The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry.We also evaluated the immunogenicity and protective efficacy of pcFlaA against V.harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test.We successfully transfected the fish muscle with pcFlaA.The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection.The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II.Furthermore,the immunized fish generated specific antibody.The vaccination also resulted in significantly higher survival during the bacterial challenge test.
基金Supported by the Higher Educational Cultivation Program for Major Scientific Research Projects of Guangdong Ocean University(GDOU2015050216)Outstanding Young Backbone Teacher Cultivation Program of Guangdong Ocean University(HDYQ2015005)+1 种基金Natural Science Foundation of Guangdong Province(2017A030313174)Guangdong Provincial Science and Technology Planning Project(2014A020208117 and 2015A020209163)
文摘[Objective] This study was conducted to determine the in-vitro inhibitory effects of Prunus mume,Coptis chinensis and Crataegus pinnatifida on Vibrio harveyi and its biofilm. [Method]The inhibitory zone diameters of the three Chinese herbal medicines against V. harveyi were determined by agar diffusion method; the minimal inhibitory concentration( MIC) and minimal bactericidal concentration( MBC) values of the three Chinese herbal medicines against V. harveyi were determined by doubling dilution method; and the effects of the three Chinese herbal medicines on the formation of V. harveyi biofilm were determined by methyl thiazolyl tetrazolium( MTT) method. [Result]The three Chinese herbal medicines all inhibited V. harveyi to different degrees. C. chinensis and C. pinnatifida and P. mume exhibited the inhibitory zone diameters of( 17. 62 ± 0. 04),( 20. 16 ± 0. 08) and( 30. 76 ± 0. 26) mm against V. harveyi,respectively. P. mume and C. pinnatifida had strong inhibitory effects on V. harveyi. The MIC and MBC values of P. mume against V. harveyi were 7. 812 5 mg/ml; the MIC and MBC values of C. pinnatifida against V. harveyi were 31. 25 mg/ml; and the MIC and MBC values of C. chinensis against V. harveyi were 62. 5 mg/ml. P. mume had the strongest antibacterial and bactericidal ability. The MIC values of C. pinnatifida,C. chinensis and P. mume against V. harveyi were 7. 81,7. 81 and 1. 96 mg/ml,respectively,i. e.,P. mume exhibited the lowest MIC. [Conclusion] P. mume,C. pinnatifida and C. chinensis all have inhibitory effects on V. harveyi and its biofilm,and P. mume has the strongest bactericidal ability.
文摘Vibrio harveyi cells (dose>103 cells mL -1) and extracellular products (ECP; >25 μg mL -1 of total protein concentration) destroyed haematopoietic cultures of Nephrops norvegicus within 24 h of exposure. Cytopathic effects (CPE) started after 4 h of exposure to the bacterial cells, with some granularity in the cytoplasm, mostly in cells in the outer periphe-ry of the explant growth. At the end of the infection, a considerable number of nuclei remained attached to the substrate, apparently unaffected. Following exposure to ECP, initial deterioration was observed at 2 h with the presence of granularity in the cytoplasm of<20% cells, and few cells displayed small vacuoles around the nuclei. Parallel results were obtained using whole animal experiments, with V. harveyi cells being lethal to nephrops within 24 h.
基金supported financially by the General Program of Natural Science Foundation of Guangdong Province(2021A1515011165)the Department of Education of Guangdong Province (2021ZDZX4005)+2 种基金the National Natural Science Foundation of China (31972808)the Research and Demonstration of Precision Functional Compound Feed Technology of Major Cultured Fishes and Shrimps in South China (2021B0202050002)the China Agriculture Research System of MOF and MARA (CARS-47)。
文摘Several reports have revealed the vital role that probiotics play in fish growth and health.However,few works are available for host gut-derived probiotics on the growth,immunity,and gut microbiota of fish,especially in hybrid grouper (♀Epinephelus fuscoguttatus×♂Epinephelus lanceolatus) due to their isolation difficulty and functional verification.This study aimed at assessing 3 host gut-derived Bacillus species?effects on the growth,immune and antioxidant-biochemical responses,haematological parameters,intestinal morphology,immune-related gene expression,gut microbiota,and disease resistance against Vibrio harveyi in hybrid grouper.A total of 480 hybrid grouper (initial weight=9.03±0.02 g) were randomly allotted into 4 groups,namely,the group fed a basal diet without probiotic inclusion (control,B0),the group fed the basal diet with Bacillus velezensis GPSAK4 (BV),the group fed the basal diet with Bacillus subtilis GPSAK9 (BS),and the group fed the basal diet with Bacillus tequilensis GPSAK2 (BT) strains at 1.0×10^(9)CFU/g.After a 6-week feeding trial,the results revealed significant improvements (P<0.05) in the growth performance,whole fish-body proximate composition,blood haematological parameters,serum,liver,and intestinal biochemical indexes,intestinal morphology,and protection against V.harveyi pathogen in the probiotic-treated groups compared with the untreated.Additionally,the expressions of intestinal tight junction genes (occludin and ZO1),pro-and anti-inflammatory genes,including IL1β,IL6,IL8,TNFa,MyD88,IL10,and TGFβ,were upregulated (P<0.05) after Bacillus species administration.Host gut-derived Bacillus supplementation shaped the gut microbiota by significantly increasing (P<0.05) the relative abundance of Proteobacteria,Bacteroidetes,Actinobacteria (except the BS group),Acidobacteria(except the BT group),Cyanobacteria (except the BV and BT groups),and Verrucomicrobia phyla,as well as known beneficial genera (Romboutsia,Turicibacter,Epulopiscium,Clostridium_sensu_stricto 1 and 13,Lactobacillus,and Bacillus),but significantly decreased (P<0.05) the abundance of Firmicutes,Chloroflexi,and Fusobacteria phyla,and purported pathogenic genera (Staphylococcus and Photobacterium) compared with the control group.Collectively,the results suggest that B.velezensis GPSAK4,B.subtilis GPSAK9(especially this strain),B.tequilensis GPSAK2 dietary supplementation at 1.0×10^(9)CFU/g has positive effects on the intestinal health of hybrid grouper via microbial composition modulation,thus enhancing the assimilation and absorption of nutrients to boost fish growth,immunity,and disease resistance.
基金the National Key Research and Development Program of the Ministry of Science and Technology(CN)(No.2022YFD2400401)the Key Research and Development Plan of Shandong Province(CN)(for Academician Team in Shandong)(No.2023ZLYS02)+1 种基金the Fundamental Research Funds for the Central Universities(No.202261029)the Enterprise Authorized Project(No.20200025)。
文摘Turbot Scophthalmus maximus is an important mariculture fish species with high economic value.However,the bacterial diseases caused by Vibrio anguillarum infection bring huge economic losses to the turbot aquaculture industry.To understand the immune response of the turbot against V.anguillarum infection and to explore novel immune-related genes,the transcriptome analysis of turbot spleen and gills were conducted after V.anguillarum infection.Differentially expressed genes(DEGs)were identified in spleen and gill of the turbot amounted to 17261 and 16436,respectively.A large number of immunerelated DEGs were enriched in cytokine-cytokine receptor interaction signaling pathway,and the others by the kyoto encyclopedia of genes and genomes(KEGG)enrichment.The gene ontology(GO)classification analysis revealed that V.anguillarum infection had the greatest effect on biological processes and cellular components.Twelve immune-related DEGs were identified in the spleen(cstl.1,egfl6,lamb21,v2rx4,calcr,and gpr78a)and gills(ghra,sh3gl2a,cst12,inhbaa,cxcl8,and il-1b)by heat map.The proteinprotein interaction(PPI)networks were constructed to analyze the immune mechanism.The results demonstrate that the maturation and antigen processing of major histocompatibility complex(MHC)class II molecule,and calcitonin-or adrenomedullin-regulated physiological activity were important events in the immunity of turbot against V.anguillarum infection.In the gills,the protein interactions in TGF-βsignaling pathway,production of inflammatory factors,and endocytosis regulation were most significant.Our research laid a foundation for discovering novel immune-related genes and enriching the knowledge of immune mechanisms of turbot against V.anguillarum infection.
基金The authors are grateful to the National High-Tech Research and Development Program of China(No.2003AA603011)Program of Science and Technology of Fujian Province(No.2002N009)for providing financial assistance for this study.
文摘Vibrio harveyi,the major causative agent of vib-riosis,affects a diverse range of marine cultured organisms over a wide geographical area.However,reports about screening the effective antigen and research on vaccines of V.harveyi are scarce.Flagellin,lipopolysaccharide(LPS)and outer membrane proteins(OMP)are major immunogenic antigens in many Gram-negative bacteria.In this study,the flagellin,OMP and LPS of the V.harveyi TS-628 strain iso-lated from infected groupers were extracted and Western blot analysis was used to detect the antigenicity of these extrac-tions.Results of the Western blot assay reveal that there are four positive flagellin bands:35 kDa,38 kDa,43 kDa,and 52 kDa,of which the 43 kDa and 52 kDa bands displayed the strongest positive reaction.There are five positive OMPbands about 35 kDa,38 kDa,43 kDa,47 kDa,and 52 kDa,of which the 43 kDa appeared to have the strongest positive reaction although the other four proteins also displayed strong reac-tions.However,LPS is Western blot-negative.These results indicate that the 43 kDa and 52 kDa flagellin and OMP of size 43 kDa,52 kDa can be candidates for developing vaccines against V.harveyi.
基金Supported by Rajiv Gandhi National Fellowship,as Junior Research Fellowship from University Grants Commission,New Delhi,Govt.of India[Grant No:F.16-1723(SC)/2010(SA-III)].
文摘Objective:To evaluate the in vitro biofilm forming capacity on abiotic food contact surfaces by Vibrio harveyi(V.harveyi)strains.Methods:Thirty six Gram-negative V.harveyi strains were isolated from various street vended seafood outlets in a food processing line and evaluated for their ability to produce mucoid biofilms on food contact surfaces using a microplate assay.Phenotypic characterization of mucoid biofilm producing V.harveyi strains were screened on Congo red agar,thiosulfate-citrate-bile salts-sucrose agar and tryptic soy agar,respectively.Results:Only five V.harveyi strains(14%)were mucoid biofilm producers characterized by formation of black colonies,whereas the remaining 31 strains(86%)were not capable of producing biofilm characterized by formation of red colonies or pinkish-red colonies with darkening at the centre.The morphological,physiological and biochemical characteristics of these isolates were studied using standard protocols.Strain identification was confirmed by polymerase chain reaction targeted to species-specific polymerase chain reaction primers VH-1 and VH-2 corresponding to variable regions of V.harveyi 16S rRNA sequence.All the biofilm-forming strains showed resistance to at least three antimicrobial compounds tested.V.harveyi strains isolated from various seafood were able to form biofilms of different capacity,and the strains VB267,VB238 and VB166 isolated from cat fish,shrimp and eel fish exhibited significantly greater biofilm forming ability compared to other isolates.Conclusions:It can be concluded from the present study that the strain VB166 was able to better attach and form subsequent biofilms on glass and stainless steel compared to high density polyethylene.These properties allow these bacteria to survive,proliferate and persist in street vended seafood outlets.
