凡纳滨对虾感染致急性肝胰腺坏死病副溶血弧菌(Vp_(AHPND))的定量分析
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摘要
对虾急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease,AHPND)是由致AHPND副溶血弧菌(AHPND-causing Vibrio parahaemolyticus,VpAHPND)携带的pVA1-like质粒所表达的PirA~(Vp)和PirB~(Vp)毒力蛋白对对虾肝胰腺的急性毒性所致。本研究用2.19×10~5 CFU/ml VpAHPND分离株20130629002S01对凡纳滨对虾(Litopenaeus vannamei)进行浸泡感染,于感染后2~9 d采集对虾的肝胰腺、鳃、肠道、肌肉组织,采用实时荧光定量PCR方法,检测各组织中的pir AVp拷贝数。结果显示,感染后凡纳滨对虾各组织均能检测到pirA~(Vp),其中,肝胰腺在感染后第4天达到峰值,为8.71×10~4 copies/mg,而鳃、肌肉、肠道分别在第3、4、5天达到峰值,分别为9.08×10~3、2.59×10~4、5.76×10~4 copies/mg。早期感染鳃组织中先出现Vp_(AHPND)的富集,在高死亡发生期,Vp_(AHPND)数量在肝胰腺和肠道出现高峰,在死亡数量逐渐下降的后期,各组织的Vp_(AHPND)均快速下降,肠道、肝胰腺和肌肉中的Vp_(AHPND)水平趋于接近。对虾肝胰腺组织病理切片显示,同一时间有临床症状的病虾和濒死对虾相比,濒死对虾表现出更严重的AHPND病理特征,且二者的组织病理特征均随着感染时间的延长变得更为严重,但检测到的Vp_(AHPND)数量呈下降趋势。研究表明,在Vp_(AHPND)感染过程中,组织中的pirA~(Vp)基因数量不能代表对虾的发病程度,发病程度及组织病理严重的AHPND样品中Vp_(AHPND)的数量不一定处于高水平状态。
Acute hepatopancreatic necrosis disease(AHPND) results from acute toxicity in the hepatopancreas of infected shrimp caused by the toxic proteins PirA~(Vp) and PirB~(Vp), which are expressed by the pVA1 plasmid carried by AHPND-causing Vibrio parahaemolyticus(Vp_(AHPND)). In this study, Litopenaeus vannamei were exposed to 2.19×10~5 CFU/ml Vp_(AHPND) strain 20130629002 S01 by immersion to explore the dynamic changes of Vp_(AHPND) in the tissues of shrimp. The hepatopancreas, gills, midgut, and muscle of infected shrimp were collected 2~9 days after immersion infection, and the quantity of pirA~(VP) was measured by qPCR. The results showed that Vp_(AHPND) could be detected in all sampled tissues of infected shrimp. The amount of Vp_(AHPND) in the hepatopancreas reached a peak on day 4 post-infection at 8.71×10~4 copies/mg, while the gills, muscle, and midgut reached peaks on day 3, 4, and 5 post-infection at 9.08×10~3、2.59×10~4、5.76×10~4 copies/mg, respectively. The highest amount of Vp_(AHPND) was detected in the gills during the early stage of infection, followed by the hepatopancreas and midgut in sequence during heavy disease, with frequent deaths. Subsequently, the amount of Vp_(AHPND) declined rapidly in all tissues, with similar levels in the midgut, hepatopancreas, and muscle. Histopathology revealed that AHPND lesions were denser in hepatopancreas samples from moribund shrimp compared with those from morbid shrimp, when taken at the same time from infection. Furthermore, the histopathologic symptoms of both became more severe along the infection process, but with decreasing levels of VpAHPND. The results showed that the copy number of pirA~(Vp) in tissues of Vp_(AHPND)-infected shrimp does not represent the real-time condition of diseased shrimp and the quantity of VpAHPND may not be high in a severe AHPND sample.
Acute hepatopancreatic necrosis disease(AHPND) results from acute toxicity in the hepatopancreas of infected shrimp caused by the toxic proteins PirA~(Vp) and PirB~(Vp), which are expressed by the pVA1 plasmid carried by AHPND-causing Vibrio parahaemolyticus(Vp_(AHPND)). In this study, Litopenaeus vannamei were exposed to 2.19×10~5 CFU/ml Vp_(AHPND) strain 20130629002 S01 by immersion to explore the dynamic changes of Vp_(AHPND) in the tissues of shrimp. The hepatopancreas, gills, midgut, and muscle of infected shrimp were collected 2~9 days after immersion infection, and the quantity of pirA~(VP) was measured by qPCR. The results showed that Vp_(AHPND) could be detected in all sampled tissues of infected shrimp. The amount of Vp_(AHPND) in the hepatopancreas reached a peak on day 4 post-infection at 8.71×10~4 copies/mg, while the gills, muscle, and midgut reached peaks on day 3, 4, and 5 post-infection at 9.08×10~3、2.59×10~4、5.76×10~4 copies/mg, respectively. The highest amount of Vp_(AHPND) was detected in the gills during the early stage of infection, followed by the hepatopancreas and midgut in sequence during heavy disease, with frequent deaths. Subsequently, the amount of Vp_(AHPND) declined rapidly in all tissues, with similar levels in the midgut, hepatopancreas, and muscle. Histopathology revealed that AHPND lesions were denser in hepatopancreas samples from moribund shrimp compared with those from morbid shrimp, when taken at the same time from infection. Furthermore, the histopathologic symptoms of both became more severe along the infection process, but with decreasing levels of VpAHPND. The results showed that the copy number of pirA~(Vp) in tissues of Vp_(AHPND)-infected shrimp does not represent the real-time condition of diseased shrimp and the quantity of VpAHPND may not be high in a severe AHPND sample.
引文
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Flegel TW.Historic emergence,impact and current status of shrimp pathogens in Asia.Journal of Invertebrate Pathology,2012,110(2):166-173
Han JE,Tang KFJ,Tran LH,et al.Photorhabdus insect-related(Pir)toxin-like genes in a plasmid of Vibrio parahaemolyticus,the causative agent of acute hepatopancreatic necrosis disease(AHPND)of shrimp.Diseases of Aquatic Organisms,2015a,113(1):33-40
Han JE,Tang KFJ,Pantoja CR,et al.q PCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease(AHPND)due to pathogenic Vibrio parahaemolyticus.Aquaculture,2015b,442:12-15
Joshi J,Srisala J,Truong VH,et al.Variation in Vibrio parahaemolyticus,isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease(AHPND).Aquaculture,2014,428-429:297-302
la Pe?a LD,Cabillon NA,Catedral DD,et al.Acute hepatopancreatic necrosis disease(AHPND)outbreaks in Penaeus vannamei and P.monodon cultured in the Philippines.Diseases of Aquatic Organisms,2015,116(3):251-254
Lee CT,Chen IT,Yang YT,et al.The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803
Lightner DV,Redman RM,Pantoja CR,et al.Early mortality syndrome affects shrimp in Asia.Global Aquaculture Advocate,2012,(Jan/Feb):40.https://www.aquaculturealliance.org/pdf/GAA-Lightner-Jan12.pdf
Nunan L,Lightner DV,Pantoja C,et al.Detection of acute hepatopancreatic necrosis disease(AHPND)in Mexico.Diseases Aquatic Organisms,2014,111(1):81-86
OIE.Manual of diagnostic tests for aquatic animals.World Organisation of Animal Health.2017,http://www.oie.int/international-standard-setting/aquatic-manual/access-online/
Otta SK,Praveena P,Bhuvaneswari T,et al.Protocols for detection of shrimp EMS/AHPND.Training Manual on Health Management Practices for Finfish and Shellfish of Brackish Water Environment 115.Aquaculture Animal Health and Environment Division,Central Institute of Brackishwater Aquaculture,Puran,Chennai,2014,98-100
Sirikharin R,Taengchaiyaphum S,Sritunyalucksana K,et al.Anew and improved PCR method for detection of AHPNDbacteria.https://enaca.org/?id=96&title=new-pcr-detectionmethod-for-ahpnd,2014
Sotorodriguez SA,Gomezgil B,Lozanoolvera R,et al.Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp(Litopenaeus vannamei)in Northwestern Mexico.Applied and Environmental Microbiology,2014,81(5):1689-1699
Tang KFJ,Pantoja CR,Redman RM,et al.Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei(EHP),a microsporidian parasite infecting penaeid shrimp.Journal of Invertebrate Pathology,2015,130:37-41
Tran L,Nunan L,Redman RM,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.Diseases of Aquatic Organisms,2013,105(1):45-55
Yang YT,Chen IT,Lee CT,et al.Draft genome sequences of four strains of Vibrio parahaemolyticus,three of which cause early mortality syndrome/acute hepatopancreatic necrosis disease in shrimp in China and Thailand.Genome Announcements,2013,2(5):814-830
Zhang BC,Liu F,Bian HH,et al.Isolation,identification,and pathogenicity analysis of a Vibrio parahaemolyticus strain from Litopenaeus vannamei.Progress in Fishery Sciences,2012,33(2):56-62[张宝存,刘飞,边慧慧,等.一株凡纳滨对虾病原菌的分离、鉴定及其致病力分析.渔业科学进展,2012,33(2):56-62]
Zhang QL,Liu Q,Liu S,et al.A new nodavirus is associated with covert mortality disease of shrimp.Journal of General Virology,2014,95(12):2700-2709
Brown WF.Variance estimation in Reed-Muench fifty percent end-point determination.American Journal of Epidemiology,1964,79(1):37-46
Fisheries and Fisheries Administration Bureau of the Ministry of Agriculture.China fishery statistics yearbook.Beijing:China Agricultural Press,2016[农业部渔业渔政管理局.中国渔业统计年鉴.北京:中国农业出版社,2016]
FAO.Report of the FAO/MARD technical workshop on early mortality syndrome(EMS)or acute hepatopancreatic necrosis syndrome(AHPNS)of cultured shrimp(under TCP/VIE/3304)Hanoi,Viet Nam,25-27 June,2013,FAOFisheries and Aquaculture Report,1053
Flegel TW.Historic emergence,impact and current status of shrimp pathogens in Asia.Journal of Invertebrate Pathology,2012,110(2):166-173
Han JE,Tang KFJ,Tran LH,et al.Photorhabdus insect-related(Pir)toxin-like genes in a plasmid of Vibrio parahaemolyticus,the causative agent of acute hepatopancreatic necrosis disease(AHPND)of shrimp.Diseases of Aquatic Organisms,2015a,113(1):33-40
Han JE,Tang KFJ,Pantoja CR,et al.q PCR assay for detecting and quantifying a virulence plasmid in acute hepatopancreatic necrosis disease(AHPND)due to pathogenic Vibrio parahaemolyticus.Aquaculture,2015b,442:12-15
Joshi J,Srisala J,Truong VH,et al.Variation in Vibrio parahaemolyticus,isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease(AHPND).Aquaculture,2014,428-429:297-302
la Pe?a LD,Cabillon NA,Catedral DD,et al.Acute hepatopancreatic necrosis disease(AHPND)outbreaks in Penaeus vannamei and P.monodon cultured in the Philippines.Diseases of Aquatic Organisms,2015,116(3):251-254
Lee CT,Chen IT,Yang YT,et al.The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin.Proceedings of the National Academy of Sciences of the United States of America,2015,112(34):10798-10803
Lightner DV,Redman RM,Pantoja CR,et al.Early mortality syndrome affects shrimp in Asia.Global Aquaculture Advocate,2012,(Jan/Feb):40.https://www.aquaculturealliance.org/pdf/GAA-Lightner-Jan12.pdf
Nunan L,Lightner DV,Pantoja C,et al.Detection of acute hepatopancreatic necrosis disease(AHPND)in Mexico.Diseases Aquatic Organisms,2014,111(1):81-86
OIE.Manual of diagnostic tests for aquatic animals.World Organisation of Animal Health.2017,http://www.oie.int/international-standard-setting/aquatic-manual/access-online/
Otta SK,Praveena P,Bhuvaneswari T,et al.Protocols for detection of shrimp EMS/AHPND.Training Manual on Health Management Practices for Finfish and Shellfish of Brackish Water Environment 115.Aquaculture Animal Health and Environment Division,Central Institute of Brackishwater Aquaculture,Puran,Chennai,2014,98-100
Sirikharin R,Taengchaiyaphum S,Sritunyalucksana K,et al.Anew and improved PCR method for detection of AHPNDbacteria.https://enaca.org/?id=96&title=new-pcr-detectionmethod-for-ahpnd,2014
Sotorodriguez SA,Gomezgil B,Lozanoolvera R,et al.Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp(Litopenaeus vannamei)in Northwestern Mexico.Applied and Environmental Microbiology,2014,81(5):1689-1699
Tang KFJ,Pantoja CR,Redman RM,et al.Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei(EHP),a microsporidian parasite infecting penaeid shrimp.Journal of Invertebrate Pathology,2015,130:37-41
Tran L,Nunan L,Redman RM,et al.Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.Diseases of Aquatic Organisms,2013,105(1):45-55
Yang YT,Chen IT,Lee CT,et al.Draft genome sequences of four strains of Vibrio parahaemolyticus,three of which cause early mortality syndrome/acute hepatopancreatic necrosis disease in shrimp in China and Thailand.Genome Announcements,2013,2(5):814-830
Zhang BC,Liu F,Bian HH,et al.Isolation,identification,and pathogenicity analysis of a Vibrio parahaemolyticus strain from Litopenaeus vannamei.Progress in Fishery Sciences,2012,33(2):56-62[张宝存,刘飞,边慧慧,等.一株凡纳滨对虾病原菌的分离、鉴定及其致病力分析.渔业科学进展,2012,33(2):56-62]
Zhang QL,Liu Q,Liu S,et al.A new nodavirus is associated with covert mortality disease of shrimp.Journal of General Virology,2014,95(12):2700-2709