Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs...Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among 14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of 〈1.0×10^5 colony forming units （CFU）mL^-1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillantm with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay （IFA） was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.展开更多
Nucleotide oligomerization domain 2(NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals.However, studies regarding NOD2, especially the initiated signal...Nucleotide oligomerization domain 2(NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals.However, studies regarding NOD2, especially the initiated signaling pathways, are scarce in teleost species. In this study, we identified a NOD2 molecule(PaNOD2) from ayu(Plecoglossus altivelis).Bioinformatics analysis showed the structure of NOD2 to be highly conserved during vertebrate evolution. Dual-luciferase reporter assays examined the activation of NF-κB signaling and Western blotting analysis detected the phosphorylation of three MAP kinases(p-38, Erk1/2, and JNK1/2).Functional study revealed that, like its mammalian counterparts, PaNOD2 was the receptor of the bacterial cell wall component muramyl dipeptide(MDP), and the leucine-rich repeat motif was responsible for the recognition and binding of Pa NOD2 with the ligand. Overexpression of PaNOD2 activated the NF-κB signaling pathway, leading to the upregulation of inflammatory cytokines, including TNF-α and IL-1β in HEK293 T cells and ayu head kidney-derived monocytes/macrophages(MO/MΦ).Particularly, we found that PaNOD2 activated the MAPK signaling pathways, as indicated by the increased phosphorylation of p-38, Erk1/2, and JNK1/2, which have not been characterized in any teleost species previously. Our findings proved that the NOD2 molecule and initiated pathways are conserved between mammals and ayu. Therefore, ayu could be used as an animal model to investigate NOD2-based diseases and therapeutic applications.展开更多
The dietary selenium (Se) requirement of yellowtail kingfish (Seriola lalandi) in normal and infected conditions was investigated. The fish were fed one of five experimental diets;a control un-supplemented diet (3.35 ...The dietary selenium (Se) requirement of yellowtail kingfish (Seriola lalandi) in normal and infected conditions was investigated. The fish were fed one of five experimental diets;a control un-supplemented diet (3.35 mg/kg Se) or diets supplemented with Se to provide 4.86, 5.38, 5.85 or 6.38 mg/kg Se. After feeding for 6 weeks, the fish were challenged by Vibrio anguillarum immersion and then observed for 4 weeks. Supplementation of Se had no effect on feed intake, feed conversion ratio and survival over 6 weeks of feeding;however, it significantly increased growth and Se content in muscle tissues. The optimal Se level for maximal growth of yellowtail kingfish estimated by second order regression was 5.56 mg/kg. Following the bacterial challenge, the immune-stimulating effects of Se were demonstrated in lysozyme and bactericidal activities, and there was a corresponding increase in survival and antibody response by supplementation of Se at ≥2 mg/kg (measured Se of ≥5.38 mg/kg). Under normal and infectious conditions, antioxidant capacity of fish measured as glutathione peroxidase activity increased by supplementation of Se. During post-challenge period, haematocrits were higher in the fish fed Se supplemented diets than the fish fed the control diet, while more macrophage aggregates were seen in the control group than in the others. Furthermore, there was evidence of myopathy in fish fed the diet without Se supplementation. Therefore, the results indicated that the optimal dietary Se requirement of yellowtail kingfish is 5.56 mg/kg.展开更多
The characteristics of a bacterium strain M3, isolated from cultured flounder Paralichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to t...The characteristics of a bacterium strain M3, isolated from cultured flounder Paralichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the species Vibrio anguillarum . Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic for Paralichthys olivacues . The LD 50 dose was 5.144×10 3 CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell free supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease.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
Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study...Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemocyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a 'U' like expression profile resulted. Expression levels of penaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimicrobial activities. In WSSV infection experiments, the expression profile appeared as an inverse 'U' with the expression of penaeidin gradually decreasing to below baseline level after 24 h. The expression of antimicrobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms of virus to haemocytes and microbial to haemocytes are completely different. Decline of penaeidins expression levels may be due to haemocytes being destroyed by WSSV or that the virus can inhibit the expression of penaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.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
Freeze-drying,continuous passage and ultra-low temperature cryopreservation are often used to preserve pathogens.In this study,Vibrio anguillarum was rejuvenated by intramuscular infection as the initial strain.The di...Freeze-drying,continuous passage and ultra-low temperature cryopreservation are often used to preserve pathogens.In this study,Vibrio anguillarum was rejuvenated by intramuscular infection as the initial strain.The difference between cells preserved with different preservation methods and their initial strains were compared with physiological and biochemical methods and through antibiotics resistance analysis.The composition of protectants for freeze-drying V.anguillarum was optimized.We found that the optimal composition of protectants was 8% of trehalose,12% of skim milk,8.0% of lactose,2.0% of sodium citrate,12.0% of serum and 8.0% of mannitol.The indexes of lysine decarboxylase and urease changed after continuous passage.The urease reaction changed after freeze-drying and freeze-thawing,but the reaction can be restored to the initial after freeze-drying.Based on the antibiotics resistance analyses,the sensitivity of V.anguillarum to different drugs including rifampicin,erythrocin,furazolidone,ceftazidime,lomefloxacin,gentamycin,azithromycin,doxycycline,ampicillin,co-trimoxazole and cefoperazone changed after different treatments,and some of these changes can be restored to the original through activation culture.In sum,compared with cryopreservation and continuous passage,the freeze-drying is more sustainable for the long-term preservation of V.anguillarum,which showed a better effect in maintaining the original characteristics of pathogen.展开更多
基金This study was supported by National High Technology Development Program of China(863,Grant 2006AA100306)the Science Foundation of Shandong Province(032070104).
文摘Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among 14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of 〈1.0×10^5 colony forming units （CFU）mL^-1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillantm with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay （IFA） was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.
基金supported by the National Natural Science Foundation of China(31772876,31702374)Natural Science Foundation of Zhejiang Province(LZ18C190001,LQ17C190001)+1 种基金Zhejiang Xinmiao Talents Program(2017R405023)K.C.Wong Magna Fund in Ningbo University
文摘Nucleotide oligomerization domain 2(NOD2) is a major cytoplasmic sensor for pathogens and is critical for the clearance of cytosolic bacteria in mammals.However, studies regarding NOD2, especially the initiated signaling pathways, are scarce in teleost species. In this study, we identified a NOD2 molecule(PaNOD2) from ayu(Plecoglossus altivelis).Bioinformatics analysis showed the structure of NOD2 to be highly conserved during vertebrate evolution. Dual-luciferase reporter assays examined the activation of NF-κB signaling and Western blotting analysis detected the phosphorylation of three MAP kinases(p-38, Erk1/2, and JNK1/2).Functional study revealed that, like its mammalian counterparts, PaNOD2 was the receptor of the bacterial cell wall component muramyl dipeptide(MDP), and the leucine-rich repeat motif was responsible for the recognition and binding of Pa NOD2 with the ligand. Overexpression of PaNOD2 activated the NF-κB signaling pathway, leading to the upregulation of inflammatory cytokines, including TNF-α and IL-1β in HEK293 T cells and ayu head kidney-derived monocytes/macrophages(MO/MΦ).Particularly, we found that PaNOD2 activated the MAPK signaling pathways, as indicated by the increased phosphorylation of p-38, Erk1/2, and JNK1/2, which have not been characterized in any teleost species previously. Our findings proved that the NOD2 molecule and initiated pathways are conserved between mammals and ayu. Therefore, ayu could be used as an animal model to investigate NOD2-based diseases and therapeutic applications.
文摘The dietary selenium (Se) requirement of yellowtail kingfish (Seriola lalandi) in normal and infected conditions was investigated. The fish were fed one of five experimental diets;a control un-supplemented diet (3.35 mg/kg Se) or diets supplemented with Se to provide 4.86, 5.38, 5.85 or 6.38 mg/kg Se. After feeding for 6 weeks, the fish were challenged by Vibrio anguillarum immersion and then observed for 4 weeks. Supplementation of Se had no effect on feed intake, feed conversion ratio and survival over 6 weeks of feeding;however, it significantly increased growth and Se content in muscle tissues. The optimal Se level for maximal growth of yellowtail kingfish estimated by second order regression was 5.56 mg/kg. Following the bacterial challenge, the immune-stimulating effects of Se were demonstrated in lysozyme and bactericidal activities, and there was a corresponding increase in survival and antibody response by supplementation of Se at ≥2 mg/kg (measured Se of ≥5.38 mg/kg). Under normal and infectious conditions, antioxidant capacity of fish measured as glutathione peroxidase activity increased by supplementation of Se. During post-challenge period, haematocrits were higher in the fish fed Se supplemented diets than the fish fed the control diet, while more macrophage aggregates were seen in the control group than in the others. Furthermore, there was evidence of myopathy in fish fed the diet without Se supplementation. Therefore, the results indicated that the optimal dietary Se requirement of yellowtail kingfish is 5.56 mg/kg.
文摘The characteristics of a bacterium strain M3, isolated from cultured flounder Paralichthys olivaceus with remarkable external sign of skin ulcer during an epizootic outbreak, indicated that the bacterium belonged to the species Vibrio anguillarum . Challenge by I.M. (intramuscular injection), bath, and oral administration with M3 showed that it was highly pathogenic for Paralichthys olivacues . The LD 50 dose was 5.144×10 3 CFU/ per fish infection by I.M. injection. Recovered inoculated bacteria from the surviving fish revealed that the asymptomatic carriers could be a latent contagious source. Study of the effect of bacterial culture CFS (cell free supernatant) showed that the exotoxins produced by M3 play an important role in its pathogenicity for flounder. The resistance of M3 to 36 out of 41 antibiotics indicated that the bacterial disease outbreak was mainly attributable to the frequent and excessive use of antimicrobial agents; and that vaccination would be an effective precaution against bacterial disease.
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Penaeidin from Chinese shrimp (Fenneropenaeus chinensis) has proved to be one of the most important antimicrobial peptides in the bodies of animals. The relative quantitative real-time PCR method is developed to study through time, the mRNA expression profile of penaeidin in the muscle and haemocyte tissue of Chinese shrimp infected with vibrio (Vibrio anguillarum) and WSSV (white spot syndrome virus). Research results showed that the same pathogens infection experiments produced similar gene expression profile in different tissues while different expression profiles appeared in the same tissues infected by different exterior pathogens. In vibrio infection experiments, a 'U' like expression profile resulted. Expression levels of penaeidin increased and surpassed the non-stimulated level, indicating that penaeidin from Chinese shrimp has noticeable antimicrobial activities. In WSSV infection experiments, the expression profile appeared as an inverse 'U' with the expression of penaeidin gradually decreasing to below baseline level after 24 h. The expression of antimicrobial peptides gene in mRNA level in response to virus infection in shrimp showed that international mechanisms of virus to haemocytes and microbial to haemocytes are completely different. Decline of penaeidins expression levels may be due to haemocytes being destroyed by WSSV or that the virus can inhibit the expression of penaeidins by yet undiscovered modes. The expression profiles of penaeidin in response to exterior pathogen and the difference of expression profiles between vibrio and WSSV infection provided some clues to further understanding the complex innate immune mechanism in shrimp.
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funded by the Key Projects of Science and Technology Innovation of Shandong Province (No. 2018YFJH0703)the Central Public-Interest Scientific Institution Basal Research Fund+1 种基金Yellow Sea Fisheries Research Institutes, CAFS (No. 20603022017004)the Projects of International Exchange and Cooperation in Agriculture, Ministry of Agriculture and Rural Affairs of China-Science, Technology and Innovation Cooperation in Aquaculture with Tropical Countries
文摘Freeze-drying,continuous passage and ultra-low temperature cryopreservation are often used to preserve pathogens.In this study,Vibrio anguillarum was rejuvenated by intramuscular infection as the initial strain.The difference between cells preserved with different preservation methods and their initial strains were compared with physiological and biochemical methods and through antibiotics resistance analysis.The composition of protectants for freeze-drying V.anguillarum was optimized.We found that the optimal composition of protectants was 8% of trehalose,12% of skim milk,8.0% of lactose,2.0% of sodium citrate,12.0% of serum and 8.0% of mannitol.The indexes of lysine decarboxylase and urease changed after continuous passage.The urease reaction changed after freeze-drying and freeze-thawing,but the reaction can be restored to the initial after freeze-drying.Based on the antibiotics resistance analyses,the sensitivity of V.anguillarum to different drugs including rifampicin,erythrocin,furazolidone,ceftazidime,lomefloxacin,gentamycin,azithromycin,doxycycline,ampicillin,co-trimoxazole and cefoperazone changed after different treatments,and some of these changes can be restored to the original through activation culture.In sum,compared with cryopreservation and continuous passage,the freeze-drying is more sustainable for the long-term preservation of V.anguillarum,which showed a better effect in maintaining the original characteristics of pathogen.
