Long non-coding RNAs(lncRNAs)are a class of transcripts longer than 200 bp,which have been emerged as essential regulators in numerous biological processes.Black rockfish(Sebastes schlegelii)is an economic fish that w...Long non-coding RNAs(lncRNAs)are a class of transcripts longer than 200 bp,which have been emerged as essential regulators in numerous biological processes.Black rockfish(Sebastes schlegelii)is an economic fish that widely cultured in the coastal areas of China,Japan,and South Korea.With the expansion of aquacultural scale,various pathogens have threatened its industry and reduced its economic values.It has been reported that lncRNA were involved in the immune response and metabolic pathway in teleost,while no study is available on identification and functional analysis of lncRNAs in black rockfish so far.Herein,this study was performed to identify lncRNAs in the intestine of black rockfish after Edwardsiella tarda infection.In our results,a total of 9311 lncRNAs were identified through highthroughput sequencing,and 102 lncRNAs were significantly regulated following challenge,which were predicted to target 3348 mRNAs.Results of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of the se target genes showed they were function in catalytic activity,hydrolase activity,defense response and peptidase activity,which involved in metabolic pathways and immune related pathways.In addition,47 lncRNAs and 8 differentially expressed mRNAs(DEmRNAs)showed co-expression at two or more infection time points with metabolism and immunity functions.Moreover,real-time quantitative PCR(qRT-PCR)was performed to verify the reliability of sequencing gene expression analysis results.This research laid the foundation for further investigation of the regulatory roles of lncRNAs in the intestinal immune response of black rockfish.展开更多
Mitogen-activated protein kinases(MAPKs)play pivotal roles in response to environmental stresses and bacterial infections.Compared with those in the higher vertebrates,studies of mapk gene family are still limited in ...Mitogen-activated protein kinases(MAPKs)play pivotal roles in response to environmental stresses and bacterial infections.Compared with those in the higher vertebrates,studies of mapk gene family are still limited in teleost.Identification,characterization,classification,and expression profiling of totally 15 mapk genes in black rockfish(Sebastes schlegelii)were conducted.Phylogenetic relationships show that these mapk genes could be divided into extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),and p38 sub-families.In addition,gene structures,syntenic analysis,and selective pressure analysis are performed to confirm their annotations.Results of selective pressure analysis indicate that mapk1,mapk3,mapk7,mapk10,mapk11,and mapk12 underwent significantly-positive selections,while the others genes such as mapk4,mapk6,mapk15,mapk8a,mapk8b,mapk9,mapk13,mapk14a,and mapk14b were under purifying selections.Moreover,results of qRT-PCR indicate that mapk genes in 8 healthy tissues displayed different expression patterns.The expression patterns of several mapk genes including mapk12,mapk13,mapk14a,mapk14b,and mapk15 were significantly changed in mucosal tissues after Edwardsiella piscicida infection.This study demonstrates that mapk genes in black rockfish play vital prevention roles against bacterial infection,which not only helps us understand the structure and function of mapk genes in black rockfish,but also provides a reference to understand the role of mapk genes in teleost immune responses.展开更多
Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In t...Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.展开更多
Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of m...Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of microbial pathogens.In this study,the gene expression profiles of TLRs in the spleen,head kidney,gill,small intestine,liver,muscle,and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR(qPCR).The TLR family members were widely expressed in different tissues with different basic expression profiles.The highest expressions of TLR1,5m,7,8,9,14,and 21 were found in the spleen;the highest expressions of TLR3 and TLR21 were found in the gill;the highest expressions of TLR2 and 5s were found in the small intestine.The second highest expressions of TLR3,7,and 8 were found in small intestine.The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA,RNA,and lipopolysaccharide(LPS)were also detected in spleen,head kidney and gill.TLR9 and TLR21 were sensitive to E.tarda DNA;TLR 8 and TLR21 were sensitive to E.tarda RNA;and TLR1 and TLR14 were sensitive to E.tarda LPS.The expressions of the other TLR genes showed no significant changes.The results imply that the expressions of these TLR genes in P.olivaceus are differently regulated in the whole body and play important roles in the immune response against E.tarda infection.展开更多
Over the past five years, an epidemic disease broke out in Pelteobagrus fulvidraco ponds of Zhejiang Province. The diseased fish mainly exhibits head splitting and surface bleeding; dissection results reveal hepatonep...Over the past five years, an epidemic disease broke out in Pelteobagrus fulvidraco ponds of Zhejiang Province. The diseased fish mainly exhibits head splitting and surface bleeding; dissection results reveal hepatonephromegaly and abdominal bleeding. A bacterial strain HSY201301 was isolated from the liver tissue of P. fulvidraco with typical symptoms. Artificial infection experiment confirmed that the isolated strain had a strong virulence to healthy P. fidvidraco, leading to similar symptoms to naturally infected P. fulvidraco. The isolated strain was identified as an Edwardsiella tarda strain according to conventional morphological, physiological, biochemical characteristics and 16S rRNA gene sequence. Results of drug susceptibility test indicated that the isolated strain was sensitive to cipro- floxacin, doxitard, penicillin, doxycycline, and rocephin. This study laid solid foundation for effective prevention and control of E. tarda.展开更多
A study was carried out to investigate antibiotic resistance patterns and plasmid profiling of Edwardsiella tarda isolated from farmed-cultured Heterobranchus longifilis in Lagos State, Southwest of Nigeria. A total o...A study was carried out to investigate antibiotic resistance patterns and plasmid profiling of Edwardsiella tarda isolated from farmed-cultured Heterobranchus longifilis in Lagos State, Southwest of Nigeria. A total of 44 Edwardsiella isolates were recovered from 80 fish samples collected from the 10 fish farms using selective random stratification. It was observed that Edwardsiella tarda isolates were 100% resistant to Amoxicillin, Chloranphenicol, Levofloxacin, Streptomycin and 90% resistant to Nalidixic Acid respectively. All the isolates were 100% susceptible to Spectinomycin and Ciprofloxacin, while Ofloxacin, Gentamycin, and Pefloxacin vary in their level of susceptibility with 90%, 80% and 70% sensitivity respectively. Conversely, 8 out of 10 fish farm locations studied were observed to have antibiotic-resistant strains, and 5 out of 8 drug-resistant strains were found to carry plasmid and the sizes of the plasmid ranges between 20.027 kb to 23.130 kb. The plasmid after treatment with mitomycin C and ethidium bromide were lost during the process of plasmid curing confirming that the multiple drug resistant exhibited by the isolates was plasmid mediated. There are fewer studies on antibiotic resistance in Edwardsiella tarda from aquaculture enterprises and this study provides further support to the view that there is a potential risk of transfer of resistant bacteria and their genes to human pathogen through the food chain. Although, in Nigeria there is no antibiotic product registered for aquaculture usage, yet fish farmers use them off-label for bacterial diseases prevention.展开更多
2013年春季,广东佛山养殖的长吻鮠Leiocassis longirostris大范围死亡。从病鱼体内分离到一株病原菌GDFS-01,通过菌落形态观察、革兰氏染色镜检及生理生化鉴定,结合16S r DNA序列测定与系统发育分析,鉴定为鮰爱德华氏菌Edwardsiella ict...2013年春季,广东佛山养殖的长吻鮠Leiocassis longirostris大范围死亡。从病鱼体内分离到一株病原菌GDFS-01,通过菌落形态观察、革兰氏染色镜检及生理生化鉴定,结合16S r DNA序列测定与系统发育分析,鉴定为鮰爱德华氏菌Edwardsiella ictaluri。采取腹部注射和浸泡感染试验均证实该菌有致病性,病症与自然发病症状一致。药敏试验表明:分离菌株对恩诺沙星、氟苯尼考、阿莫西林、洛美沙星、多西环素、磺胺甲基异唑、土霉素等7种药物敏感,对青霉素、氨苄西林、氟哌酸、四环素、万古霉素、利福平、克林霉素等药物呈耐药。本研究结果为长吻鮠病害的有效防控提供参考。展开更多
Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) wit...Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB-LAMP). In this method, the Mg 2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene (hemolysin-LAMP) for detection of E. tarda. The EsrB-LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.展开更多
Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgent...Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgently.To achieve this purpose,we developed a TaqMan-based real-time PCR assay for detection and quantification of E.tarda.The assay targets the hemolysin activator HlyB domain protein of E.tarda.Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction.A standard curve was generated from the threshold cycle values(y) against log10(E.tarda genomic DNA concentration) as x.The intra-and inter-assay coefficient of variation(CV) values were less than 2.06% and 1.05% respectively,indicating that the assay had good reproducibility.This method is highly specific to E.tarda strains,as it shows no cross-reactivity to Edwardsiella ictaluri,a member of the same genus,or to nine other fish-pathogenic bacteria species belonging to three other genera.This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E.tarda in clinical samples.展开更多
基金Supported by the Young Experts of Taishan Scholars(No.tsqn201909130)the Science and Technology Support Plan for Youth Innovation of Colleges and Universities in Shandong Province(No.2019KJF003)+1 种基金the“First Class Fishery Discipline”Program in Shandong Province,a special talent program“One Matter One Decision(Yi Shi Yi Yi)”Program in Shandong Province,Chinathe Breeding Plan of Shandong Provincial Qingchuang Research Team(2019)。
文摘Long non-coding RNAs(lncRNAs)are a class of transcripts longer than 200 bp,which have been emerged as essential regulators in numerous biological processes.Black rockfish(Sebastes schlegelii)is an economic fish that widely cultured in the coastal areas of China,Japan,and South Korea.With the expansion of aquacultural scale,various pathogens have threatened its industry and reduced its economic values.It has been reported that lncRNA were involved in the immune response and metabolic pathway in teleost,while no study is available on identification and functional analysis of lncRNAs in black rockfish so far.Herein,this study was performed to identify lncRNAs in the intestine of black rockfish after Edwardsiella tarda infection.In our results,a total of 9311 lncRNAs were identified through highthroughput sequencing,and 102 lncRNAs were significantly regulated following challenge,which were predicted to target 3348 mRNAs.Results of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses of the se target genes showed they were function in catalytic activity,hydrolase activity,defense response and peptidase activity,which involved in metabolic pathways and immune related pathways.In addition,47 lncRNAs and 8 differentially expressed mRNAs(DEmRNAs)showed co-expression at two or more infection time points with metabolism and immunity functions.Moreover,real-time quantitative PCR(qRT-PCR)was performed to verify the reliability of sequencing gene expression analysis results.This research laid the foundation for further investigation of the regulatory roles of lncRNAs in the intestinal immune response of black rockfish.
基金Supported by the National Key R&D Program of China(No.2018YFD0900101)the Young Experts of Taishan Scholars(No.tsqn201909130)+2 种基金the Science and Technology Support Plan for Youth Innovation of Colleges and Universities in Shandong Province(No.2019KJF003)the“First Class Fishery Discipline”Program in Shandong Provincethe Shandong Technical System of Fish Industry(No.SDAIT-12-03)。
文摘Mitogen-activated protein kinases(MAPKs)play pivotal roles in response to environmental stresses and bacterial infections.Compared with those in the higher vertebrates,studies of mapk gene family are still limited in teleost.Identification,characterization,classification,and expression profiling of totally 15 mapk genes in black rockfish(Sebastes schlegelii)were conducted.Phylogenetic relationships show that these mapk genes could be divided into extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),and p38 sub-families.In addition,gene structures,syntenic analysis,and selective pressure analysis are performed to confirm their annotations.Results of selective pressure analysis indicate that mapk1,mapk3,mapk7,mapk10,mapk11,and mapk12 underwent significantly-positive selections,while the others genes such as mapk4,mapk6,mapk15,mapk8a,mapk8b,mapk9,mapk13,mapk14a,and mapk14b were under purifying selections.Moreover,results of qRT-PCR indicate that mapk genes in 8 healthy tissues displayed different expression patterns.The expression patterns of several mapk genes including mapk12,mapk13,mapk14a,mapk14b,and mapk15 were significantly changed in mucosal tissues after Edwardsiella piscicida infection.This study demonstrates that mapk genes in black rockfish play vital prevention roles against bacterial infection,which not only helps us understand the structure and function of mapk genes in black rockfish,but also provides a reference to understand the role of mapk genes in teleost immune responses.
基金The Special Fund for Agro-scientific Research in the Public Interest under contract No.201103034Construction Special Fund of Modern Agriculture and Industrial Technology Research System under contract No.CARS-47
文摘Edwardsiella tarda is one of the most important emerging pathogens in tile global aquaculture industries. As such, an accurate diagnosis and quantitative analytical methods are urgently needed for this bacterium. In this study, primers and a TaqMan probe specific to the conservative sequences of the 16S rRNA gene of E. tarda were designed. The concentration of primers and TaqMan probe were optimized to 200 nmol/L and 120 nmol/L, respectively. The detection sensitivity of the FQ- PCR assay was determined to be as low as five copies of the target sequence per reaction using the pGEM-16S rDNA recombinant plasmid as a template, which was 100 times more sensitive than conventional PCR. A standard curve by plotting the threshold cycle values （y） against the common logarithmic copies （logl0n~ as x; n~ is copy number） of pGEM-16S rDNA was generated. The results of intra- and inter-assay variability tests demonstrate that the established FQ-PCR method was highly reproducible. The assay was specific for E. tarda as it showed that there was no cross-reactivity to eight additional bacterial pathogen strains in aquaculture. Thus, the FQ-PCR assay has the potential for diagnostic purposes and for other applications, especially for the rapid detection and quantification of low-grade E. tarda infections.
基金supported by the Applied Basic Research Programs of the Science and Technology Commission Foundation of Tianjin, China (Nos. 19JCZDJC34300, 14JCZDJC34200 and 18JCYBJC96100)
文摘Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of microbial pathogens.In this study,the gene expression profiles of TLRs in the spleen,head kidney,gill,small intestine,liver,muscle,and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR(qPCR).The TLR family members were widely expressed in different tissues with different basic expression profiles.The highest expressions of TLR1,5m,7,8,9,14,and 21 were found in the spleen;the highest expressions of TLR3 and TLR21 were found in the gill;the highest expressions of TLR2 and 5s were found in the small intestine.The second highest expressions of TLR3,7,and 8 were found in small intestine.The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA,RNA,and lipopolysaccharide(LPS)were also detected in spleen,head kidney and gill.TLR9 and TLR21 were sensitive to E.tarda DNA;TLR 8 and TLR21 were sensitive to E.tarda RNA;and TLR1 and TLR14 were sensitive to E.tarda LPS.The expressions of the other TLR genes showed no significant changes.The results imply that the expressions of these TLR genes in P.olivaceus are differently regulated in the whole body and play important roles in the immune response against E.tarda infection.
文摘Over the past five years, an epidemic disease broke out in Pelteobagrus fulvidraco ponds of Zhejiang Province. The diseased fish mainly exhibits head splitting and surface bleeding; dissection results reveal hepatonephromegaly and abdominal bleeding. A bacterial strain HSY201301 was isolated from the liver tissue of P. fulvidraco with typical symptoms. Artificial infection experiment confirmed that the isolated strain had a strong virulence to healthy P. fidvidraco, leading to similar symptoms to naturally infected P. fulvidraco. The isolated strain was identified as an Edwardsiella tarda strain according to conventional morphological, physiological, biochemical characteristics and 16S rRNA gene sequence. Results of drug susceptibility test indicated that the isolated strain was sensitive to cipro- floxacin, doxitard, penicillin, doxycycline, and rocephin. This study laid solid foundation for effective prevention and control of E. tarda.
文摘A study was carried out to investigate antibiotic resistance patterns and plasmid profiling of Edwardsiella tarda isolated from farmed-cultured Heterobranchus longifilis in Lagos State, Southwest of Nigeria. A total of 44 Edwardsiella isolates were recovered from 80 fish samples collected from the 10 fish farms using selective random stratification. It was observed that Edwardsiella tarda isolates were 100% resistant to Amoxicillin, Chloranphenicol, Levofloxacin, Streptomycin and 90% resistant to Nalidixic Acid respectively. All the isolates were 100% susceptible to Spectinomycin and Ciprofloxacin, while Ofloxacin, Gentamycin, and Pefloxacin vary in their level of susceptibility with 90%, 80% and 70% sensitivity respectively. Conversely, 8 out of 10 fish farm locations studied were observed to have antibiotic-resistant strains, and 5 out of 8 drug-resistant strains were found to carry plasmid and the sizes of the plasmid ranges between 20.027 kb to 23.130 kb. The plasmid after treatment with mitomycin C and ethidium bromide were lost during the process of plasmid curing confirming that the multiple drug resistant exhibited by the isolates was plasmid mediated. There are fewer studies on antibiotic resistance in Edwardsiella tarda from aquaculture enterprises and this study provides further support to the view that there is a potential risk of transfer of resistant bacteria and their genes to human pathogen through the food chain. Although, in Nigeria there is no antibiotic product registered for aquaculture usage, yet fish farmers use them off-label for bacterial diseases prevention.
文摘2013年春季,广东佛山养殖的长吻鮠Leiocassis longirostris大范围死亡。从病鱼体内分离到一株病原菌GDFS-01,通过菌落形态观察、革兰氏染色镜检及生理生化鉴定,结合16S r DNA序列测定与系统发育分析,鉴定为鮰爱德华氏菌Edwardsiella ictaluri。采取腹部注射和浸泡感染试验均证实该菌有致病性,病症与自然发病症状一致。药敏试验表明:分离菌株对恩诺沙星、氟苯尼考、阿莫西林、洛美沙星、多西环素、磺胺甲基异唑、土霉素等7种药物敏感,对青霉素、氨苄西林、氟哌酸、四环素、万古霉素、利福平、克林霉素等药物呈耐药。本研究结果为长吻鮠病害的有效防控提供参考。
基金Supported by the Special Fund for Agro-Scientific Research in the Public Interest (No. 201103034)the Earmarked Fund for Modern Agroindustry Technology Research System (No. nycytx-46)
文摘Edwardsiella tarda is a major pathogen in aquatic environments that can cause heavy economic losses. An improved method for quick and accurate detection of E. tarda by loop-mediated isothermal amplification (LAMP) with two additional loop primers was developed by targeting the EsrB gene (EsrB-LAMP). In this method, the Mg 2+ concentration, reaction temperature, and reaction time were optimized to 8 mmol/L, 61°C, and 40 min, respectively. The detection limit with the EsrB gene was as low as 10 copies, which is 100 times more sensitive than that of conventional polymerase chain reaction (PCR). The EsrB-LAMP assay was shown more sensitive and rapid than previously reported LAMP assays targeting the hemolysin gene (hemolysin-LAMP) for detection of E. tarda. The EsrB-LAMP was also highly specific to E. tarda and had no cross-reaction with 13 other strains of bacteria. The assay can be carried out in a simple heating device and the EsrB-LAMP products can be visually detected by adding fluorescent dye to the reaction mixture. Taken together, the improved EsrB-LAMP diagnostic protocol has the potential for detection of E. tarda from indoor and outdoor samples.
基金Supported by the Special Fund for Agro-scientific Research in the Public Interest(No.201103034)the Construction Special Fund of Modern Agriculture and Industrial Technology Research System(No.CARS-47)
文摘Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgently.To achieve this purpose,we developed a TaqMan-based real-time PCR assay for detection and quantification of E.tarda.The assay targets the hemolysin activator HlyB domain protein of E.tarda.Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction.A standard curve was generated from the threshold cycle values(y) against log10(E.tarda genomic DNA concentration) as x.The intra-and inter-assay coefficient of variation(CV) values were less than 2.06% and 1.05% respectively,indicating that the assay had good reproducibility.This method is highly specific to E.tarda strains,as it shows no cross-reactivity to Edwardsiella ictaluri,a member of the same genus,or to nine other fish-pathogenic bacteria species belonging to three other genera.This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E.tarda in clinical samples.
