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
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
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
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
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
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
Vibrio parahaemolyticus is the leading causal agent of human acute gas- troenteritis. Real-time accurate detection means is the key to prevention and control of its spread. This study provided a novel detection strate...Vibrio parahaemolyticus is the leading causal agent of human acute gas- troenteritis. Real-time accurate detection means is the key to prevention and control of its spread. This study provided a novel detection strategy for realizing rapid and specific determination of V. parahaemolyticus by labeling its monoclonal antibody （Ab） with quantum dots （QDs）. The results showed that the fluorescence of these QDs-Ab bioconjugates was quenched by graphene oxide （GO） to produce a bacteri- um capture probe. And the optimal quenched concentration of GO was 60 ng/ml. When the bacterium capture probe was exposed to the target, green color fluores- cence was turned on by releasing the QDs-Ab due to the antibody antigen combi- nation. The detection limit of V. parahaemolyticus was 104 CFU/ml based on 3 times signal-to-noise ratio. The specificity of the FRET sensor towards V. para- haemolyticus was examined by comparing with controls such as V. splendidus, V. alginolyticus, Edwardsiella tarda and Aeromonas hydrophila with the same condition. The controls couldn＇t cause obvious fluorescence alteration, while the target resulted in significant fluorescence enhancement. This strategy could be further used as a universal method for any bacterial determination by changing the conjugated antibod- ies in early disease diagnosis. Therefore, the sensor has good potential to expand its application to the early diagnosis and determination of bacteria.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
In order to explore the inhibitory effects of Prunus mume,Crataegus pinnatifida and Coptis chinensis on Vibrio parahaemolyticus and its biofilm in vitro,the agar diffusion method was applied. These three Chinese herba...In order to explore the inhibitory effects of Prunus mume,Crataegus pinnatifida and Coptis chinensis on Vibrio parahaemolyticus and its biofilm in vitro,the agar diffusion method was applied. These three Chinese herbal medicines had different inhibitory effects on V. parahaemolyticus. The inhibition zone of C. pinnatifida to V. parahaemolyticus was( 15. 25 ± 0. 53) mm,and the minimum inhibitory concentration( MIC) and minimum bactericidal concentration( MBC) of C. pinnatifida on V. parahaemolyticus were both 31. 25 mg/m L; the inhibition zone of C. chinensis to V. parahaemolyticus was( 18. 08 ± 0. 10) mm,and the MIC and MBC of C. chinensis on V. parahaemolyticus were both15. 63 mg/m L; the inhibition zone of P. mume to V. parahaemolyticus was( 28. 99 ± 0. 47) mm,and the MIC and MBC of P. mume on V. parahaemolyticus were both 7. 81 mg/m L. The effects of three traditional Chinese medicines on the biofilm formation of V. parahaemolyticus were tested by MTT colorimetric method using methylthiazolyl tetrazolium( MTT). P. mume,C. pinnatifida and C. chinensis have significant inhibitory effects on V. parahaemolyticus biofilm and their MIC are 7. 81 mg/m L,3. 125 mg/m L,and 62. 5 mg/m L,respectively( P < 0. 01).The experimental results are expected to provide certain references for the development of new fishery drugs.展开更多
Objectives:Vibrio parahaemolyticus is the primary species that causes vibriosis.In this study,a point-of-care detection method was developed for V.parahaemolyticus.Materials and Methods:The detection platform targeted...Objectives:Vibrio parahaemolyticus is the primary species that causes vibriosis.In this study,a point-of-care detection method was developed for V.parahaemolyticus.Materials and Methods:The detection platform targeted the thermolabile haemolysin(tlh)gene of V.parahaemolyticus based on recombinant polymerase amplifcation(RPA)and clustered regularly spaced short palindromic repeat(CRISPR/Cas)systems.The platform was combined with an immunochromatographic test strip(ICS)that enables low-cost,simple,visual detection of V.parahaemolyticus.Results:The detection limit was 2.5×10^(2)fg/µL for plasmids and 1.4×10^(2)CFU/mL for V.parahaemolyticus.In addition,V.parahaemolyticus in salmon sashimi could be detected at a concentration of 154 CFU/g without enrichment,and the entire detection time was around 30 min.After enrichment for 6 h,2 CFU/g V.parahaemolyticus could be detected.Conclusions:Consequently,the proposed RPA-CRISPR/Cas12a-ICS platform could detect V.parahaemolyticus in seafood intuitively,quickly,and sensitively,leading to high practical application value.展开更多
This study aimed to evaluate the supplementation of the probiotic Pseudoalteromonas piscicida 1Ub to the biofloc system as an ecofriendly strategy for protecting white shrimp(Penaeus vannamei)from Vibrio parahaemolyti...This study aimed to evaluate the supplementation of the probiotic Pseudoalteromonas piscicida 1Ub to the biofloc system as an ecofriendly strategy for protecting white shrimp(Penaeus vannamei)from Vibrio parahaemolyticus infection.Shrimp with an average body weight of(0.50±0.09)g were reared in 30 glass jars with a working volume of 2.5 L at a density of 20 ind/L.Shrimp were reared for 5 d for each treatment,which included the biofloc system without and with 106 colony forming unit(CFU)per mL probiotic.The regular clear water system was used as control.All treatment groups were challenged with 103,105,and 107 CFU/mL V.parahaemolyticus.For the negative control,shrimp were reared without V.parahaemolyticus.The results showed that the density of V.parahaemolyticus cocultured with P.piscicida 1Ub decreased and the density of V.parahaemolyticus in rearing water and shrimp body in the probiotic-treated group was lower than that in the control group(P<0.05).The survival and immune response(total hemocyte count,phagocytic activity,respiratory burst,phenoloxidase,and superoxide dismutase)of shrimp in the probiotic group was higher than that in the positive control(P<0.05).Moreover,supplementing the biofloc system with the probiotic could protect shrimp hepatopancreas from damage caused by V.parahaemolyticus,regardless of bacterial density.Thus,the supplementation of the probiotic P.piscicida 1Ub in the biofloc system could significantly protect and increase the resistance of shrimp to V.parahaemolyticus infection.展开更多
Background:Swift and accurate detection of Vibrio parahaemolyticus,which is a prominent causative pathogen associated with seafood contamination,is required to effectively combat foodborne disease and wound infections...Background:Swift and accurate detection of Vibrio parahaemolyticus,which is a prominent causative pathogen associated with seafood contamination,is required to effectively combat foodborne disease and wound infections.The toxR gene is relatively conserved within V.parahaemolyticus and is primarily involved in the expression and regulation of virulence genes with a notable degree of specificity.The aim of this study was to develop a rapid,simple,and constant temperature detection method for V.parahaemolyticus in clinical and nonspecialized laboratory settings.Methods:In this study,specific primers and CRISPR RNA were used to target the toxR gene to construct a reaction system that combines recombinase polymerase amplification(RPA)with CRISPR‒Cas13a.The whole-genome DNA of the sample was extracted by self-prepared sodium dodecyl sulphate(SDS)nucleic acid rapid extraction reagent,and visual interpretation of the detection results was performed by lateral flow dipsticks(LFDs).Results:The specificity of the RPA-CRISPR/Cas13a-LFD method was validated using V.parahaemolyticus strain ATCC-17802 and six other non-parahaemolytic Vibrio species.The results demonstrated a specificity of 100%.Additionally,the genomic DNA of V.parahaemolyticus was serially diluted and analysed,with a minimum detectable limit of 1 copy/μL for this method,which was greater than that of the TaqMan-qPCR method(10^(2) copies/μL).The established methods were successfully applied to detect wild-type V.parahaemolyticus,yielding results consistent with those of TaqMan-qPCR and MALDI-TOF MS mass spectrometry identification.Finally,the established RPA-CRISPR/Cas13a-LFD method was applied to whole blood specimens from mice infected with V.parahaemolyticus,and the detection rate of V.parahaemolyticus by this method was consistent with that of the conventional PCR method.Conclusions:In this study,we describe an RPA-CRISPR/Cas13a detection method that specifically targets the toxR gene and offers advantages such as simplicity,rapidity,high specificity,and visual interpretation.This method serves as a valuable tool for the prompt detection of V.parahaemolyticus in nonspecialized laboratory settings.展开更多
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 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.
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 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.
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Supported by Shandong Scientific and Technological Development Program(2014GHY115024)~~
文摘Vibrio parahaemolyticus is the leading causal agent of human acute gas- troenteritis. Real-time accurate detection means is the key to prevention and control of its spread. This study provided a novel detection strategy for realizing rapid and specific determination of V. parahaemolyticus by labeling its monoclonal antibody （Ab） with quantum dots （QDs）. The results showed that the fluorescence of these QDs-Ab bioconjugates was quenched by graphene oxide （GO） to produce a bacteri- um capture probe. And the optimal quenched concentration of GO was 60 ng/ml. When the bacterium capture probe was exposed to the target, green color fluores- cence was turned on by releasing the QDs-Ab due to the antibody antigen combi- nation. The detection limit of V. parahaemolyticus was 104 CFU/ml based on 3 times signal-to-noise ratio. The specificity of the FRET sensor towards V. para- haemolyticus was examined by comparing with controls such as V. splendidus, V. alginolyticus, Edwardsiella tarda and Aeromonas hydrophila with the same condition. The controls couldn＇t cause obvious fluorescence alteration, while the target resulted in significant fluorescence enhancement. This strategy could be further used as a universal method for any bacterial determination by changing the conjugated antibod- ies in early disease diagnosis. Therefore, the sensor has good potential to expand its application to the early diagnosis and determination of bacteria.
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 National Natural Science Foundation of Guangdong Province(2017A030313174)Natural Science Foundation of Guangdong Ocean University(C17379)+1 种基金Undergraduate Innovative and Entrepreneurial Team Project(CCTD201802)Special Project for Fishing Port Construction and Fishery Industry Development of Guangdong Province(A201708A05)
文摘In order to explore the inhibitory effects of Prunus mume,Crataegus pinnatifida and Coptis chinensis on Vibrio parahaemolyticus and its biofilm in vitro,the agar diffusion method was applied. These three Chinese herbal medicines had different inhibitory effects on V. parahaemolyticus. The inhibition zone of C. pinnatifida to V. parahaemolyticus was( 15. 25 ± 0. 53) mm,and the minimum inhibitory concentration( MIC) and minimum bactericidal concentration( MBC) of C. pinnatifida on V. parahaemolyticus were both 31. 25 mg/m L; the inhibition zone of C. chinensis to V. parahaemolyticus was( 18. 08 ± 0. 10) mm,and the MIC and MBC of C. chinensis on V. parahaemolyticus were both15. 63 mg/m L; the inhibition zone of P. mume to V. parahaemolyticus was( 28. 99 ± 0. 47) mm,and the MIC and MBC of P. mume on V. parahaemolyticus were both 7. 81 mg/m L. The effects of three traditional Chinese medicines on the biofilm formation of V. parahaemolyticus were tested by MTT colorimetric method using methylthiazolyl tetrazolium( MTT). P. mume,C. pinnatifida and C. chinensis have significant inhibitory effects on V. parahaemolyticus biofilm and their MIC are 7. 81 mg/m L,3. 125 mg/m L,and 62. 5 mg/m L,respectively( P < 0. 01).The experimental results are expected to provide certain references for the development of new fishery drugs.
基金supported by the Scientifc and Innovative Action Plan of Shanghai(No.21N31900800)the Shanghai Rising-Star Program(No.23QB1403500)+2 种基金the Shanghai Science and Technology Commission,the Belt and Road Project(No.20310750500)the Talent Project of SAAS(No.2023–2025)the SAAS Program for Excellent Research Team(No.2022(B-16)),China.
文摘Objectives:Vibrio parahaemolyticus is the primary species that causes vibriosis.In this study,a point-of-care detection method was developed for V.parahaemolyticus.Materials and Methods:The detection platform targeted the thermolabile haemolysin(tlh)gene of V.parahaemolyticus based on recombinant polymerase amplifcation(RPA)and clustered regularly spaced short palindromic repeat(CRISPR/Cas)systems.The platform was combined with an immunochromatographic test strip(ICS)that enables low-cost,simple,visual detection of V.parahaemolyticus.Results:The detection limit was 2.5×10^(2)fg/µL for plasmids and 1.4×10^(2)CFU/mL for V.parahaemolyticus.In addition,V.parahaemolyticus in salmon sashimi could be detected at a concentration of 154 CFU/g without enrichment,and the entire detection time was around 30 min.After enrichment for 6 h,2 CFU/g V.parahaemolyticus could be detected.Conclusions:Consequently,the proposed RPA-CRISPR/Cas12a-ICS platform could detect V.parahaemolyticus in seafood intuitively,quickly,and sensitively,leading to high practical application value.
基金financially supported by the Ministry of Education,Culture,Research,and Technology of the Republic of Indonesia for funding the present study in the form of the Pendidikan Magister menuju Doktor untuk Sarjana Unggul(PMDSU)program scholarship(Grant number:200/SP2H/PMDSU/DRPM/2020).
文摘This study aimed to evaluate the supplementation of the probiotic Pseudoalteromonas piscicida 1Ub to the biofloc system as an ecofriendly strategy for protecting white shrimp(Penaeus vannamei)from Vibrio parahaemolyticus infection.Shrimp with an average body weight of(0.50±0.09)g were reared in 30 glass jars with a working volume of 2.5 L at a density of 20 ind/L.Shrimp were reared for 5 d for each treatment,which included the biofloc system without and with 106 colony forming unit(CFU)per mL probiotic.The regular clear water system was used as control.All treatment groups were challenged with 103,105,and 107 CFU/mL V.parahaemolyticus.For the negative control,shrimp were reared without V.parahaemolyticus.The results showed that the density of V.parahaemolyticus cocultured with P.piscicida 1Ub decreased and the density of V.parahaemolyticus in rearing water and shrimp body in the probiotic-treated group was lower than that in the control group(P<0.05).The survival and immune response(total hemocyte count,phagocytic activity,respiratory burst,phenoloxidase,and superoxide dismutase)of shrimp in the probiotic group was higher than that in the positive control(P<0.05).Moreover,supplementing the biofloc system with the probiotic could protect shrimp hepatopancreas from damage caused by V.parahaemolyticus,regardless of bacterial density.Thus,the supplementation of the probiotic P.piscicida 1Ub in the biofloc system could significantly protect and increase the resistance of shrimp to V.parahaemolyticus infection.
文摘Background:Swift and accurate detection of Vibrio parahaemolyticus,which is a prominent causative pathogen associated with seafood contamination,is required to effectively combat foodborne disease and wound infections.The toxR gene is relatively conserved within V.parahaemolyticus and is primarily involved in the expression and regulation of virulence genes with a notable degree of specificity.The aim of this study was to develop a rapid,simple,and constant temperature detection method for V.parahaemolyticus in clinical and nonspecialized laboratory settings.Methods:In this study,specific primers and CRISPR RNA were used to target the toxR gene to construct a reaction system that combines recombinase polymerase amplification(RPA)with CRISPR‒Cas13a.The whole-genome DNA of the sample was extracted by self-prepared sodium dodecyl sulphate(SDS)nucleic acid rapid extraction reagent,and visual interpretation of the detection results was performed by lateral flow dipsticks(LFDs).Results:The specificity of the RPA-CRISPR/Cas13a-LFD method was validated using V.parahaemolyticus strain ATCC-17802 and six other non-parahaemolytic Vibrio species.The results demonstrated a specificity of 100%.Additionally,the genomic DNA of V.parahaemolyticus was serially diluted and analysed,with a minimum detectable limit of 1 copy/μL for this method,which was greater than that of the TaqMan-qPCR method(10^(2) copies/μL).The established methods were successfully applied to detect wild-type V.parahaemolyticus,yielding results consistent with those of TaqMan-qPCR and MALDI-TOF MS mass spectrometry identification.Finally,the established RPA-CRISPR/Cas13a-LFD method was applied to whole blood specimens from mice infected with V.parahaemolyticus,and the detection rate of V.parahaemolyticus by this method was consistent with that of the conventional PCR method.Conclusions:In this study,we describe an RPA-CRISPR/Cas13a detection method that specifically targets the toxR gene and offers advantages such as simplicity,rapidity,high specificity,and visual interpretation.This method serves as a valuable tool for the prompt detection of V.parahaemolyticus in nonspecialized laboratory settings.
