Although Vibrio parahaemolyticus has become the most common pathogen in fresh and frozen seafood,its virulence changes have often been ignored during the processing of seafood.To investigate these potential risks,we u...Although Vibrio parahaemolyticus has become the most common pathogen in fresh and frozen seafood,its virulence changes have often been ignored during the processing of seafood.To investigate these potential risks,we used frozen Penaeus chinensis as examples,and the most virulent factors of V.parahaemolyticus,including amounts,viable but nonculturable(VBNC)status,toxins TDH and TRH,and virulence genes tdh and trh,were determined.Bacterial quantities were signifi cantly reduced during drain and sprinkling phases,but caused by different factors.By SYTO9 and PI staining showed that washing was the main reason for the bacterial reduction at the drain phase,while the strain entering VBNC state was another reason at sprinkling phase.Their hemolysis toxicity,produced by TDH and TRH,became stronger after inoculation on shrimp,and could be detected throughout the process.Moreover,tdh and trh also exhibited trends similar to that of the hemolysis toxicity test.tdh was almost to a two-fold expression level during ice-glazing phase,while trh only express at a low level,less than half of the expression level before inoculation.These results demonstrated that the strains were not dead during freezing process,but became VBNC cells,which still produced and accumulated toxins,especially TDH,the most virulent factor.展开更多
Objective:To investigate the contamination and distribution of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou City.Methods:Three types of seafood sold in Haikou from 2020 to 2022 were collected,...Objective:To investigate the contamination and distribution of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou City.Methods:Three types of seafood sold in Haikou from 2020 to 2022 were collected,Vibrio parahaemolyticus and Vibrio alginolyticus were detected according to the National Food Safety Standard Food Microbiological Examination of Vibrio parahaemolyticus(GB 4789.7-2013),and they were identified by real-time fluorescence PCR.The detection of Vibrio parahaemolyticus and Vibrio alginolyticus in different kinds of seafood,different years and different quarters was analyzed.Results:A total of 119 seafood samples were collected.Among them,24 samples were positive with Vibrio parahaemolyticus,with a positive rate of 20.1%;46 samples were positive with Vibrio alginolyticus,with a positive rate of 38.7%.Among various types of seafood,shrimp have the highest positivity rate for Vibrio parahaemolyticus at 50%,while shellfish have the highest positivity rate for Vibrio alginolyticus at 48%.Comparing between monitoring years,the positive rate of Vibrio alginolyticus was the highest in 2021(76.7%),while the positive rate of Vibrio parahaemolyticus was the highest in 2022(25%).Comparing between different quarters,the positivity rate for Vibrio alginolyticus was found to be highest in the second quarter at 80%,while the positivity rate for Vibrio parahaemolyticus was highest in the fourth quarter at 33.3%.There were statistically significant differences(P<0.05)in the positivity rate for Vibrio alginolyticus in different years and quarters,as well as in the positivity rate for Vibrio parahaemolyticus in different types and quarters.Conclusion:Vibrio parahaemolyticus and Vibrio alginolyticus were found in seafood products in Haikou City from 2020 to 2022.It is recommended that relevant departments strengthen supervision to ensure the safety of seafood products consumed by the public.展开更多
Objective:To identify a potential bacterium which produces antimicrobial peptide(vibriocin),and its purification,characterization and production optimization.The bacteria subjected in the study were isolated from a hi...Objective:To identify a potential bacterium which produces antimicrobial peptide(vibriocin),and its purification,characterization and production optimization.The bacteria subjected in the study were isolated from a highly competitive ecological niche of mangrove ecosystem.Methods:The bacterium was characterized by phenotype besides 16S rRNA gene sequence analysis.The antibacterial activity was recognised by using agar well diffusion method.The vibriocin was purified using ammonium sulphate precipitation,butanol extraction,gel filtration chromatography,ion-exchange chromatography and subsequently,by HPLC.Molecular weight of the substance identified in SDS-PAGE.Production optimization performed according to Taguchi's mathematical model using 6 different nutritional parameters as variables.Results:The objective bacterium was identified as Vibrio parahaemolyticm.The vibriocin showed 18 KDa of molecular mass with mono peptide in nature and highest activity against pathogenic Vibrio harveyi.The peptide act stable in a wide range of pH,temperature.UV radiation,solvents and chemicals utilized.An overall^20%of vibriocin production was improved,and was noticed that NaCl and agitation speed played a vital role in secretion of vibriocin.Conclusion:The vibriocin identified here would he an effective alternative for chemically synthesized drugs for the management of Vibrio infections in mariculture industry.展开更多
Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly int...Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.展开更多
Vibrio parahaemolyticus, the leading cause of seafood-borne gastroenteritis, has the ability to form biofilms on biotic and abiotic surfaces. Biofilm formation is a complicated process involving many specific structur...Vibrio parahaemolyticus, the leading cause of seafood-borne gastroenteritis, has the ability to form biofilms on biotic and abiotic surfaces. Biofilm formation is a complicated process involving many specific structures and regulatory processes. The most significant of the structures and processes include polar and lateral flagella, mannose-sensitive hemagglutinin typeⅣpili, chitin-regulated pili,capsular polysaccharide (CPS), exopolysaccharide展开更多
The invasive red alga Grateloupia turuturu Yamada could turn Vibrio parahaemolyticus into nonculturable state in live algal culture.In order to elucidate the mechanism of such an effect,a series of culture experiments...The invasive red alga Grateloupia turuturu Yamada could turn Vibrio parahaemolyticus into nonculturable state in live algal culture.In order to elucidate the mechanism of such an effect,a series of culture experiments were performed in this investigation based on three hypothesized causes,namely bacterial attachment,production of reactive oxygen species （ROS） and the discharge of water soluble secondary metabolic compounds.The results reveal that attachment to the thallus surface of G.turuturu was the major reason for the decrease of V.parahaemolyticus in seawater.Further investigations show that V.parahaemolyticus attachment to the surface of algal thallus in live cultures of seaweeds was a common phenomenon.However,the disappearance of the culturability of V.parahaemolyticus occurred only on the thallus of G.turuturu over 72 h among all six algal species tested.Electron microscopic scanning shows that most of V.parahaemolyticus attached to G.turuturu changed from the initial normal bacilli to coccoid-shape after 72 h.The enclosure experiments by enclosing the algal thallus in tubes demonstrate that the nonculturability of V.parahaemolyticus in the water of live culture of G.turuturu occurred after the physical contact of the V.parahaemolyticus to the alga.The capacity of G.turuturu in affecting the culturability of V.parahaemolyticus was not influenced after inhibition of photosynthesis by treatment of 3（3,4dichlorophenyl）-1 ,1dimethyl urea （DCMU） at non-lethal levels.Production of reactive oxygen species after addition of live culture of bacteria was excluded by on-line analyzing the oxidation of dichlorohydrofluorescein （DCFH） to dichlorofluorescein （DCF） in the presence of peroxidase on a VersaFluor fluorometer.展开更多
White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacter...White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P <0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.展开更多
Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced b...Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.展开更多
Objective This study aimed to investigate the regulation of histone-like nucleoid structuring protein(H-NS)on biofilm formation and cyclic diguanylate(c-di-GMP)synthesis in Vibrio parahaemolyticus RIMD2210633.Methods ...Objective This study aimed to investigate the regulation of histone-like nucleoid structuring protein(H-NS)on biofilm formation and cyclic diguanylate(c-di-GMP)synthesis in Vibrio parahaemolyticus RIMD2210633.Methods Regulatory mechanisms were analyzed by the combined utilization of crystal violet staining,quantification of c-di-GMP,quantitative real-time polymerase chain reaction,LacZ fusion,and electrophoretic-mobility shift assay.Results The deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V.parahaemolyticus RIMD2210633.H-NS can bind the upstream promoter-proximal DNA regions of scrA,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979 to repress their transcription.These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism.Conclusion One of the mechanisms by which H-NS represses the biofilm formation by V.parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.展开更多
BACKGROUND Vibrio pararhaemolyticus(V.parahaemolyticus),a pathogen that commonly causes gastroenteritis,could potentially lead to a pandemic in Asia.Its pathogenesis and molecular mechanisms vary,and the severity of i...BACKGROUND Vibrio pararhaemolyticus(V.parahaemolyticus),a pathogen that commonly causes gastroenteritis,could potentially lead to a pandemic in Asia.Its pathogenesis and molecular mechanisms vary,and the severity of illness can be diverse,ranging from mild gastroenteritis,requiring only supportive care,to sepsis.CASE SUMMARY We outline a case of a 71-year-old female who experienced an acute onset of severe abdominal tenderness after two days of vomiting and diarrhea prior to her emergency department visit.A small bowel perforation was diagnosed using computed tomography.The ascites cultured revealed infection due to V.parahaemolyticus CONCLUSION Our case is the first reported case of V.parahaemolyticus-induced gastroenteritis resulting in small bowel perforation.展开更多
The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins...The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins are an ideal target for vaccines.We amplified the complete flagellin subunit gene(flaA) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21(DE3) cells.The gene coded for a protein that was 62.78 kDa.We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus.In addition,the purified FlaA protein can be used for further functional and structural studies.展开更多
Vibrio parahaemolyticus is the main cause of foodborne gastroenteritis,which is widely distributed in shellfish and other seafood.Most V.parahaemolyticus are nonpathogenic,and only a few types,such as serotype O3:K6,a...Vibrio parahaemolyticus is the main cause of foodborne gastroenteritis,which is widely distributed in shellfish and other seafood.Most V.parahaemolyticus are nonpathogenic,and only a few types,such as serotype O3:K6,are pathogenic,which is also the most prevalent strain in Asia.However,the relationship between this serotype and pathogenicity has yet to be established.The waaf gene is located in the O antigen synthesis gene cluster.Thus,we constructed a waaf gene deletion mutant(i.e.,Δwaaf)of wild-type(WT)which isolated from shellfish serotype O3:K6 via chitin-mediated transformation technology.We then constructed theΔwaaf complementary strain(i.e.,C-△waaf)via the Escherichia coli S17λpir strain by conjugation.The basic physiological characteristics,adhesion to Caco2 cells,and pathogenicity of the WT,△waaf,and C-△waaf strains were compared.Growth curves showed no remarkable differences between the WT andΔwaaf strains.However,theΔwaaf strain non-reactive to O3 antisera and other 12 O-group antisera of V.parahaemolyticus.Moreover,the number of flgella and extracellular polysaccharides decreased,the adhesion decreased,and the pathogenicity weakened.These characteristics of the C-△waaf strain were similar to those of the WT strain These results indicated that the waaf gene is vital to the serotype in V.parahaemolyticus,and changes in O antigen could affect the pathogenicity of this bacterium.This study will be helpful to understand the pathogenic mechanism of V.parahaemolyticus.展开更多
基金funded by grants from the National Key Research and Development Program of China[2018YFC1602201]Key Research and Development Program of Shaanxi Province[2021NY-158]+1 种基金the National Natural Science Foundation of China[31671780]the Agricultural Product Quality and Safety Risk Assessment Foundation of the Ministry of Agriculture and Rural Affairs of the People’s Republic of China[GJFP2020002,GJFP2020003].
文摘Although Vibrio parahaemolyticus has become the most common pathogen in fresh and frozen seafood,its virulence changes have often been ignored during the processing of seafood.To investigate these potential risks,we used frozen Penaeus chinensis as examples,and the most virulent factors of V.parahaemolyticus,including amounts,viable but nonculturable(VBNC)status,toxins TDH and TRH,and virulence genes tdh and trh,were determined.Bacterial quantities were signifi cantly reduced during drain and sprinkling phases,but caused by different factors.By SYTO9 and PI staining showed that washing was the main reason for the bacterial reduction at the drain phase,while the strain entering VBNC state was another reason at sprinkling phase.Their hemolysis toxicity,produced by TDH and TRH,became stronger after inoculation on shrimp,and could be detected throughout the process.Moreover,tdh and trh also exhibited trends similar to that of the hemolysis toxicity test.tdh was almost to a two-fold expression level during ice-glazing phase,while trh only express at a low level,less than half of the expression level before inoculation.These results demonstrated that the strains were not dead during freezing process,but became VBNC cells,which still produced and accumulated toxins,especially TDH,the most virulent factor.
基金Major Science and Technology Project of Hainan Province(No.ZDKJ202003)。
文摘Objective:To investigate the contamination and distribution of Vibrio parahaemolyticus and Vibrio alginolyticus in seafood in Haikou City.Methods:Three types of seafood sold in Haikou from 2020 to 2022 were collected,Vibrio parahaemolyticus and Vibrio alginolyticus were detected according to the National Food Safety Standard Food Microbiological Examination of Vibrio parahaemolyticus(GB 4789.7-2013),and they were identified by real-time fluorescence PCR.The detection of Vibrio parahaemolyticus and Vibrio alginolyticus in different kinds of seafood,different years and different quarters was analyzed.Results:A total of 119 seafood samples were collected.Among them,24 samples were positive with Vibrio parahaemolyticus,with a positive rate of 20.1%;46 samples were positive with Vibrio alginolyticus,with a positive rate of 38.7%.Among various types of seafood,shrimp have the highest positivity rate for Vibrio parahaemolyticus at 50%,while shellfish have the highest positivity rate for Vibrio alginolyticus at 48%.Comparing between monitoring years,the positive rate of Vibrio alginolyticus was the highest in 2021(76.7%),while the positive rate of Vibrio parahaemolyticus was the highest in 2022(25%).Comparing between different quarters,the positivity rate for Vibrio alginolyticus was found to be highest in the second quarter at 80%,while the positivity rate for Vibrio parahaemolyticus was highest in the fourth quarter at 33.3%.There were statistically significant differences(P<0.05)in the positivity rate for Vibrio alginolyticus in different years and quarters,as well as in the positivity rate for Vibrio parahaemolyticus in different types and quarters.Conclusion:Vibrio parahaemolyticus and Vibrio alginolyticus were found in seafood products in Haikou City from 2020 to 2022.It is recommended that relevant departments strengthen supervision to ensure the safety of seafood products consumed by the public.
基金Supported by the project Drug from Sea,funded by Ministry of Earth Sciences,Govt.of India(Grant#34030020005)
文摘Objective:To identify a potential bacterium which produces antimicrobial peptide(vibriocin),and its purification,characterization and production optimization.The bacteria subjected in the study were isolated from a highly competitive ecological niche of mangrove ecosystem.Methods:The bacterium was characterized by phenotype besides 16S rRNA gene sequence analysis.The antibacterial activity was recognised by using agar well diffusion method.The vibriocin was purified using ammonium sulphate precipitation,butanol extraction,gel filtration chromatography,ion-exchange chromatography and subsequently,by HPLC.Molecular weight of the substance identified in SDS-PAGE.Production optimization performed according to Taguchi's mathematical model using 6 different nutritional parameters as variables.Results:The objective bacterium was identified as Vibrio parahaemolyticm.The vibriocin showed 18 KDa of molecular mass with mono peptide in nature and highest activity against pathogenic Vibrio harveyi.The peptide act stable in a wide range of pH,temperature.UV radiation,solvents and chemicals utilized.An overall^20%of vibriocin production was improved,and was noticed that NaCl and agitation speed played a vital role in secretion of vibriocin.Conclusion:The vibriocin identified here would he an effective alternative for chemically synthesized drugs for the management of Vibrio infections in mariculture industry.
基金supported by the National Key Research and Development Plan of China[2018YFC1602500]the Natural Science Foundation of Tianjin[19JCZDJC39900]
文摘Objective To establish an ultra-sensitive,ultra-fast,visible detection method for Vibrio parahaemolyticus(VP).Methods We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a(CRISPR/Cas12a)combined with recombinase polymerase amplification and visual detection(CRISPR/Cas12a-VD).Results CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10^(-18)M(single molecule detection)within 30 min without cross-reactivity against other bacteria.When detecting pure cultures of VP,the consistency of results reached 100%compared with real-time PCR.The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10^(2)CFU/g.Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods,such as real-time PCR,and has great potential for preventing the spread of pathogens.
基金supported by the National Natural Science Foundation of China [81601809]the Natural Science Foundation of Jiangsu Province [BK20160505]
文摘Vibrio parahaemolyticus, the leading cause of seafood-borne gastroenteritis, has the ability to form biofilms on biotic and abiotic surfaces. Biofilm formation is a complicated process involving many specific structures and regulatory processes. The most significant of the structures and processes include polar and lateral flagella, mannose-sensitive hemagglutinin typeⅣpili, chitin-regulated pili,capsular polysaccharide (CPS), exopolysaccharide
基金The National Natural Science Foundation of China under contract No.30671596
文摘The invasive red alga Grateloupia turuturu Yamada could turn Vibrio parahaemolyticus into nonculturable state in live algal culture.In order to elucidate the mechanism of such an effect,a series of culture experiments were performed in this investigation based on three hypothesized causes,namely bacterial attachment,production of reactive oxygen species （ROS） and the discharge of water soluble secondary metabolic compounds.The results reveal that attachment to the thallus surface of G.turuturu was the major reason for the decrease of V.parahaemolyticus in seawater.Further investigations show that V.parahaemolyticus attachment to the surface of algal thallus in live cultures of seaweeds was a common phenomenon.However,the disappearance of the culturability of V.parahaemolyticus occurred only on the thallus of G.turuturu over 72 h among all six algal species tested.Electron microscopic scanning shows that most of V.parahaemolyticus attached to G.turuturu changed from the initial normal bacilli to coccoid-shape after 72 h.The enclosure experiments by enclosing the algal thallus in tubes demonstrate that the nonculturability of V.parahaemolyticus in the water of live culture of G.turuturu occurred after the physical contact of the V.parahaemolyticus to the alga.The capacity of G.turuturu in affecting the culturability of V.parahaemolyticus was not influenced after inhibition of photosynthesis by treatment of 3（3,4dichlorophenyl）-1 ,1dimethyl urea （DCMU） at non-lethal levels.Production of reactive oxygen species after addition of live culture of bacteria was excluded by on-line analyzing the oxidation of dichlorohydrofluorescein （DCFH） to dichlorofluorescein （DCF） in the presence of peroxidase on a VersaFluor fluorometer.
基金Supported by the National Basic Research Program of China(973 Program)(No.2012CB114405)the Special Foundation Under the Construction Program for the“Taishan Scholarship”of Shandong Province of Chinathe Program for Chinese Outstanding Talents in Agricultural Scientific Research
文摘White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P <0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.
基金supported by National High Technology Research and Development Program of China grant(2006AA1003062006AA100307)
文摘Vibrio parahaemolyticus (VP) is one of the pathogenic vibrios endangering net-cage cultured Pseudosciaena crocea,Fennerpenaeus chinensis, and shellfish in coastal areas of China. Several types of hemolysins produced by Vp have been characterized as major virulence factors.They are thermostable direct hemolysin (TDH),TDH-related hemolysin (TRH) and thermolabile hemolysin (TLH). In this study, we cloned tdh, trh, and tlh genes from the genome DNA of VP by polymerase chain reaction (PCR).We ligated the three genes into prokaryotic expression vector pET-28a (+),and transformed the recombinant plasmids into Es-cherichia coli BL21 (DE3). The expression of recombinant proteins was induced by isopropyl-β-D-thiogalacto-pyranoside (IPTG). The recombinant proteins were expressed in a form of inclusion bodies and thus purified with Ni-NTA affinity chromatography. Western blotting results showed that recombinant proteins,TDH, TRH and TLH, could be recognized by rabbit anti-VP serum. The three purified proteins were renatured by gradient dialysis.The renatured proteins exhibited hemolytic activity except for TLH in the presence of phosphatidylcholine. These results not only are helpful for better understanding these genes' functions under a single factor level, but also provide evidence for VP vaccine engineering.
基金supported by grants from the National Natural Science Foundation of China[Grant No.82072239]。
文摘Objective This study aimed to investigate the regulation of histone-like nucleoid structuring protein(H-NS)on biofilm formation and cyclic diguanylate(c-di-GMP)synthesis in Vibrio parahaemolyticus RIMD2210633.Methods Regulatory mechanisms were analyzed by the combined utilization of crystal violet staining,quantification of c-di-GMP,quantitative real-time polymerase chain reaction,LacZ fusion,and electrophoretic-mobility shift assay.Results The deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V.parahaemolyticus RIMD2210633.H-NS can bind the upstream promoter-proximal DNA regions of scrA,scrG,VP0117,VPA0198,VPA1176,VP0699,and VP2979 to repress their transcription.These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism.Conclusion One of the mechanisms by which H-NS represses the biofilm formation by V.parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.
文摘BACKGROUND Vibrio pararhaemolyticus(V.parahaemolyticus),a pathogen that commonly causes gastroenteritis,could potentially lead to a pandemic in Asia.Its pathogenesis and molecular mechanisms vary,and the severity of illness can be diverse,ranging from mild gastroenteritis,requiring only supportive care,to sepsis.CASE SUMMARY We outline a case of a 71-year-old female who experienced an acute onset of severe abdominal tenderness after two days of vomiting and diarrhea prior to her emergency department visit.A small bowel perforation was diagnosed using computed tomography.The ascites cultured revealed infection due to V.parahaemolyticus CONCLUSION Our case is the first reported case of V.parahaemolyticus-induced gastroenteritis resulting in small bowel perforation.
基金Supported by the Dalian Municipal Government of China (No. 2007B11NC069)the Key Laboratory Foundation of the Educational Department of Liaoning Province (No.2009S024)the Grant of Dalian Fisheries University (No. SY2007005)
文摘The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals.Bacteria flagellins play an important role during infection and induction of the host immune response.Thus,flagellin proteins are an ideal target for vaccines.We amplified the complete flagellin subunit gene(flaA) from V.parahaemolyticus ATCC 17802.We then cloned and expressed the gene into Escherichia coli BL21(DE3) cells.The gene coded for a protein that was 62.78 kDa.We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting,respectively.Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus.In addition,the purified FlaA protein can be used for further functional and structural studies.
基金supported by grants from the Natural Science Foundation of Chongqing(cstc2020jcyj-msxm X0685)the Science and Technology Research Program of Chongqing Municipal Education Commission(KJQN202001231)+1 种基金the National Natural Science Foundation of China(NSFC)(No.31201372)Science and Technology Project of Wanzhou in 2020。
文摘Vibrio parahaemolyticus is the main cause of foodborne gastroenteritis,which is widely distributed in shellfish and other seafood.Most V.parahaemolyticus are nonpathogenic,and only a few types,such as serotype O3:K6,are pathogenic,which is also the most prevalent strain in Asia.However,the relationship between this serotype and pathogenicity has yet to be established.The waaf gene is located in the O antigen synthesis gene cluster.Thus,we constructed a waaf gene deletion mutant(i.e.,Δwaaf)of wild-type(WT)which isolated from shellfish serotype O3:K6 via chitin-mediated transformation technology.We then constructed theΔwaaf complementary strain(i.e.,C-△waaf)via the Escherichia coli S17λpir strain by conjugation.The basic physiological characteristics,adhesion to Caco2 cells,and pathogenicity of the WT,△waaf,and C-△waaf strains were compared.Growth curves showed no remarkable differences between the WT andΔwaaf strains.However,theΔwaaf strain non-reactive to O3 antisera and other 12 O-group antisera of V.parahaemolyticus.Moreover,the number of flgella and extracellular polysaccharides decreased,the adhesion decreased,and the pathogenicity weakened.These characteristics of the C-△waaf strain were similar to those of the WT strain These results indicated that the waaf gene is vital to the serotype in V.parahaemolyticus,and changes in O antigen could affect the pathogenicity of this bacterium.This study will be helpful to understand the pathogenic mechanism of V.parahaemolyticus.
