[Objectives] This study aimed to analyze the biological activity and immunogenicity of extracellular products from Streptococcus iniae .[Methods] S. iniae was incubated with brain heart infusion agar medium （BHIA ＋ ...[Objectives] This study aimed to analyze the biological activity and immunogenicity of extracellular products from Streptococcus iniae .[Methods] S. iniae was incubated with brain heart infusion agar medium （BHIA ＋ 4% calf serum） for 60 h. The bacterial liquid was rinsed with PBS, centrifuged, and filtered through microporous filtering film to collect extracellular products （ECPs）.[Results] Extracellular proteinase （ECPase） of S. iniae exhibited amylase, protease, lecitinase, gelatinase, lipase activities and hemolytic activity but had no urease activity. EDTA, DTT and PMSF could reduce ECPase activity to 72.4%, 77.6% and 72.4%, respectively. Cu^2＋ , Ca 2＋ , K^＋ and Mg^2＋ exhibited an inhibitory effect on ECPase activity, whereas Fe^3＋ , Co^2＋ and Mn^2＋ could activate ECPase activity. ECPs had good heat stability and exhibited relatively high activities under alkaline conditions. The optimal temperature for ECPs was 55 ℃. Two-dimensional electrophoresis was performed to analyze the main protein of ECPs. The results indicated that there are 12 main bands of ECPs, and the molecular weights mainly ranged between 28-68 kDa. About 120 protein spots were detected, and the molecular weights mainly ranged between 26-95 kDa. The mouse anti- S. iniae was used for Western-blot analysis of ECPs, and the results showed that there were four proteins, with molecular weights of 26, 37, 95, and 97 kDa, respectively. The pathogenicity assay indicated that ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.[Conclusions] This study provided a certain theoretical basis for revealing the pathogenic mechanism of Streptococcus iniae .展开更多
Extracellular products (ECP) produced by Vibrio anguillarum strain M3 originally isolated from diseased flounder ( Paralichthys olivaceus ) were prepared. ECP of M3 showed gelatinase, casinase, amylase and haemolytic ...Extracellular products (ECP) produced by Vibrio anguillarum strain M3 originally isolated from diseased flounder ( Paralichthys olivaceus ) were prepared. ECP of M3 showed gelatinase, casinase, amylase and haemolytic activity on agarose plates. High protease activity against azocasin was detected. Bacterium M3 showed highest growth and protease activity at 25℃. The protease present in ECP showed maximal activity at pH 8 and 55℃; was completely inactivated by application of 80℃ heat for 30 min; was completely inhibited by EDTA and HgCl 2, and was partially inhibited by PMSF, SDS, MnCl 2 and iodoacetic acid; but not inhibited by CaCl 2 and MgCl 2. The ECP was toxic to flounder fish at LD 50 values of 3.1 μg protein /g body weight. The addition of HgCl 2 and application of heat at 50℃ decreased the lethal toxicity of ECP. When heated at 100℃, ECP lethality to flounder was completely inhibited. After intramuscular injection of ECP into flounder, it showed evident histopathological changes including necrosis of muscle, extensive deposition of haemosiderin in the spleen, dilated blood vessels congested with numerous lymphocytes in the liver. These results showed that ECP protease was a lethal factor produced by the bacterium V. anguillarum M3.展开更多
Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder...Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Westem blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5-6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards （＂marker＂） so that the alterations ofprotease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10^4 cell/mL in the indirect ELISA, while 10^5 cell/mL in the dot-ELISA.展开更多
This study was aimed to isolate sinapine-degrading bacteria from the intestinal tract of laying hens and to identify the predominant bacteria. Thirty-week old healthy laying hens were killed, and the chyme in the dige...This study was aimed to isolate sinapine-degrading bacteria from the intestinal tract of laying hens and to identify the predominant bacteria. Thirty-week old healthy laying hens were killed, and the chyme in the digestive tract was inoculated into modified Czapek medium containing sinapin and cultivated at 37 ℃for 10 days. The optical density(OD) values of the bacterial solutions at different cultivating times were detected by a spectrophotometric method. The predominant strains were identified by 16 S rRNA gene analysis. We extracted the extracellular products of the predominant strains to determine the total protein using the Coomassie brilliant blue method, and to determine the activities of some extracellular enzymes using the agar plate diffusion method. Nine strains were isolated from the lower intestinal tract of laying hens. Among the 9 strains, 5 were from the ileum, 2 were from the ceca and 2 were from the jejunum. We could not isolate any strains from the upper intestinal tract, such as the stomach and duodenum. Eight of those 9 isolated strains were gram negative and one was gram positive. Strains YD-1 and YD-2 were better than other strains in their abilities to degrade sinapine. Strains YD-1 and YD-2 were identified as Escherichia coli and Klebsiella spp., respectively, by the 16 S rRNA sequence analysis.The total protein level of the extracellular products was 1.213 g/L for YD-1 and 1.990 g/L for YD-2. Both extracellular products of YD-1 and YD-2 had the activities of protease, amylase and urease. This study confirmed that the primary site of sinapine degradation is in the lower intestinal tract of laying hens. The sinapine-degrading strains are mainly gram negative. Strains YD-1 and YD-2 are predominant in degrading sinapine and they belong to E. coli and Klebsiella spp., respectively. Both extracellular products of YD-1 and YD-2 contain protease, amylase and urease. Strain YD-2 is better than strain YD-1 in its ability to degrade sinapine.展开更多
Extracellular polymeric substances（EPS） are organic metabolic compounds excreted by microorganisms. They largely impact microbial aggregate structures and functions.Extracellular polysaccharides（EP） in EPS are res...Extracellular polymeric substances（EPS） are organic metabolic compounds excreted by microorganisms. They largely impact microbial aggregate structures and functions.Extracellular polysaccharides（EP） in EPS are responsible for the formation of microbial aggregates. In this work, we successfully separated and characterized EP from EPS of the bacterium Bacillus megaterium TF10. Extraction of EP from EPS was optimized using Sevag＇s reagent. Chemical characteristics, functional groups, and molecular weight（MW） distribution of EP were compared with the harvested EPS and soluble microbial products（SMP）. We found that the polymers of lower MW and free proteins were successfully removed by Sevag＇s reagent. The higher MW components of EPS were predominantly polysaccharides,while the polymers of lower MW tended to secrete to the supernatant and were described as SMP. A part of the proteins in the EP was polysaccharide-bonded. Our results can be further used in elucidating the complex flocculation mechanisms in which EP play a major role.展开更多
基金Supported by Natural Science Foundation of Guangdong Province(2017A030313174)Natural Science Foundation of Guangdong Ocean University(C17379)+2 种基金Undergraduate Innovative and Entrepreneurial Team Project(CCTD201802)Science and Technology Program of Guangdong Province(2015A020209163)Special Fund for Construction of Fishery Port and Development of Fishery Industry of Guangdong Province(A201708A05)
文摘[Objectives] This study aimed to analyze the biological activity and immunogenicity of extracellular products from Streptococcus iniae .[Methods] S. iniae was incubated with brain heart infusion agar medium （BHIA ＋ 4% calf serum） for 60 h. The bacterial liquid was rinsed with PBS, centrifuged, and filtered through microporous filtering film to collect extracellular products （ECPs）.[Results] Extracellular proteinase （ECPase） of S. iniae exhibited amylase, protease, lecitinase, gelatinase, lipase activities and hemolytic activity but had no urease activity. EDTA, DTT and PMSF could reduce ECPase activity to 72.4%, 77.6% and 72.4%, respectively. Cu^2＋ , Ca 2＋ , K^＋ and Mg^2＋ exhibited an inhibitory effect on ECPase activity, whereas Fe^3＋ , Co^2＋ and Mn^2＋ could activate ECPase activity. ECPs had good heat stability and exhibited relatively high activities under alkaline conditions. The optimal temperature for ECPs was 55 ℃. Two-dimensional electrophoresis was performed to analyze the main protein of ECPs. The results indicated that there are 12 main bands of ECPs, and the molecular weights mainly ranged between 28-68 kDa. About 120 protein spots were detected, and the molecular weights mainly ranged between 26-95 kDa. The mouse anti- S. iniae was used for Western-blot analysis of ECPs, and the results showed that there were four proteins, with molecular weights of 26, 37, 95, and 97 kDa, respectively. The pathogenicity assay indicated that ECPs of S. iniae were highly pathogenic to tilapia. The mortality rate of tilapia was enhanced as the concentration of ECPs increased.[Conclusions] This study provided a certain theoretical basis for revealing the pathogenic mechanism of Streptococcus iniae .
文摘Extracellular products (ECP) produced by Vibrio anguillarum strain M3 originally isolated from diseased flounder ( Paralichthys olivaceus ) were prepared. ECP of M3 showed gelatinase, casinase, amylase and haemolytic activity on agarose plates. High protease activity against azocasin was detected. Bacterium M3 showed highest growth and protease activity at 25℃. The protease present in ECP showed maximal activity at pH 8 and 55℃; was completely inactivated by application of 80℃ heat for 30 min; was completely inhibited by EDTA and HgCl 2, and was partially inhibited by PMSF, SDS, MnCl 2 and iodoacetic acid; but not inhibited by CaCl 2 and MgCl 2. The ECP was toxic to flounder fish at LD 50 values of 3.1 μg protein /g body weight. The addition of HgCl 2 and application of heat at 50℃ decreased the lethal toxicity of ECP. When heated at 100℃, ECP lethality to flounder was completely inhibited. After intramuscular injection of ECP into flounder, it showed evident histopathological changes including necrosis of muscle, extensive deposition of haemosiderin in the spleen, dilated blood vessels congested with numerous lymphocytes in the liver. These results showed that ECP protease was a lethal factor produced by the bacterium V. anguillarum M3.
文摘Dot enzyme-linked immunosorbent assay （dot-ELISA）, indirect ELISA and Westem blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Westem blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5-6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards （＂marker＂） so that the alterations ofprotease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10^4 cell/mL in the indirect ELISA, while 10^5 cell/mL in the dot-ELISA.
基金financially supported by the Basal Fund of Scientific Research Institution for Public Welfare in Sichuan Province (SASA2013A09SASA2013B09)
文摘This study was aimed to isolate sinapine-degrading bacteria from the intestinal tract of laying hens and to identify the predominant bacteria. Thirty-week old healthy laying hens were killed, and the chyme in the digestive tract was inoculated into modified Czapek medium containing sinapin and cultivated at 37 ℃for 10 days. The optical density(OD) values of the bacterial solutions at different cultivating times were detected by a spectrophotometric method. The predominant strains were identified by 16 S rRNA gene analysis. We extracted the extracellular products of the predominant strains to determine the total protein using the Coomassie brilliant blue method, and to determine the activities of some extracellular enzymes using the agar plate diffusion method. Nine strains were isolated from the lower intestinal tract of laying hens. Among the 9 strains, 5 were from the ileum, 2 were from the ceca and 2 were from the jejunum. We could not isolate any strains from the upper intestinal tract, such as the stomach and duodenum. Eight of those 9 isolated strains were gram negative and one was gram positive. Strains YD-1 and YD-2 were better than other strains in their abilities to degrade sinapine. Strains YD-1 and YD-2 were identified as Escherichia coli and Klebsiella spp., respectively, by the 16 S rRNA sequence analysis.The total protein level of the extracellular products was 1.213 g/L for YD-1 and 1.990 g/L for YD-2. Both extracellular products of YD-1 and YD-2 had the activities of protease, amylase and urease. This study confirmed that the primary site of sinapine degradation is in the lower intestinal tract of laying hens. The sinapine-degrading strains are mainly gram negative. Strains YD-1 and YD-2 are predominant in degrading sinapine and they belong to E. coli and Klebsiella spp., respectively. Both extracellular products of YD-1 and YD-2 contain protease, amylase and urease. Strain YD-2 is better than strain YD-1 in its ability to degrade sinapine.
基金supported by the National Natural Science Foundation of China (No. 21607031)Science and Technology Planning Project of Guangdong Province, China (Nos. 2014A010107023, 2015B020230002, and 2016A010103020)
文摘Extracellular polymeric substances（EPS） are organic metabolic compounds excreted by microorganisms. They largely impact microbial aggregate structures and functions.Extracellular polysaccharides（EP） in EPS are responsible for the formation of microbial aggregates. In this work, we successfully separated and characterized EP from EPS of the bacterium Bacillus megaterium TF10. Extraction of EP from EPS was optimized using Sevag＇s reagent. Chemical characteristics, functional groups, and molecular weight（MW） distribution of EP were compared with the harvested EPS and soluble microbial products（SMP）. We found that the polymers of lower MW and free proteins were successfully removed by Sevag＇s reagent. The higher MW components of EPS were predominantly polysaccharides,while the polymers of lower MW tended to secrete to the supernatant and were described as SMP. A part of the proteins in the EP was polysaccharide-bonded. Our results can be further used in elucidating the complex flocculation mechanisms in which EP play a major role.
