Application Of Real-time PCR In Detection Of Pathogens VP_AHPND And SHIV Of Litopenaeus Vannamei

Litopenaeus vannamei is an important aquaculture species in the aquaculture industry,resently,Litopenaeus vannamei culture develop rapidly in coastal areas of China and have achieved the factorization and industrialization farming mode.With the expansion of the scale of Litopenaeus vannamei breeding and the development of high-density intensive farming mode,the aquaculture water environment gradually deteriorated,a variety of shrimp diseases frequently occur and new shrimp pathogens are also found.AHPND is a new shrimp disease also an important shrimp diseases in recent years,Litopenaeus vannamei infected by AHPND can cause high mortality and led to significant economic losses to the shrimp industry.Vibrio parahaemolyticus carrying a ⁷0kbp plasmid p VA1 with toxic genes pirA^VP and pirB_VP(VP_AHPND)is the pathogen of acute hepatopancreatic necrosis disease?AHPND?.In this study,Litopenaeus vannamei were exposed to 2.19×10⁵ CFU/mL VP_AHPND strain 20130629002S01 by immersion,to explore the dynamic changes of VP_AHPND represented with pirA^VP in tissues.Hepatopancreas,gills,middle gut,and muscle of infected shrimp were collected respectively and measured the quantity of VP_AHPND by q PCR.The results showed that VP_AHPND can be detected in all of sampled tissues of infected shrimp.The highest amount of VP_AHPND was detected in hepatopancreas on day 4 post-infection at 8.71×10⁴ copies/mg tissue,followed by that in midgut?at 9.95×10³ copies/mg tissue on day 5 post-infection?,muscle?at 2.59×10⁴ copies/mg tissue on day 4 post-infection?,and gills?at 9.08×10³ copies/mg tissue on day 3 post-infection?.Sequentially,VP_AHPND firstly enriched in gills before disease,and followed in hepatopancrease and midgut in sequence during heavy disease with frequent deaths,and then VP_AHPND amount declined rapidly in all tissues and with close levels in midgut,hepatopancrease,and muscle.Samples taken at the same time after challenge showed that heavier AHPND lesions,such as sloughed tubular epithelial cells and infiltration of hemocytes,in the hepatopancreas of moribund shrimp than that of morbid shrimp.VP_AHPND amount tended to decrease at the later period of infection associated with increasing of a mass of secondary infection in hepatopancreatic tubules of moribund shrimp,which implied that the detection of PirVP representing of VP_AHPND may not result in a high level positive within severe AHPND samples.Severe mortaliities occured in a shrimp farms of Zhejiang taizhou in 2014,all of the the known common shrimp pathogens showed negative results by PCR detection.Our lab achieved a new shrimp pathogen isolate from the infected shrimps,confirmed the disease belong to Iridoviridae by the electron microscope observation and the second generation sequencing analysis,temporarily named Shrimp hemocyte iridescent virus,SHIV.In this study,we designed of a pair of primers and Taq Man probe according to SHIV hypothesis ATPase sequence to carry out quantitative detection for SHIV.The Taq Man real-time fluorescent quantitative PCR method established in this study showed a strong specificity to SHIV,the SHIV detection sensitivity was four copies SHIV/u L DNA,and the coeffcient of regression of the standard curve was 0.99.A total of 205 DNA samples extracted from farmed shrimp collected from different shrimp farms of China were tested by the real time PCR assay.Results showed that there were 19.51% SHIV-positives,these 205 clinical shrimp included 181 individuals of Litopenaeus vannamei,18 individuals of Fenneropenaeus chinensis,and 6 individuals of Macrobrachium rosenbergii.The SHIV-positive rates in different species are 16.57%,27.78%,and 83.33%,respectively.There were 30 SHIV-positive samples in 181 Litopenaeus vannamei,and the SHIV quantity were range 7.65×10¹ to 4.70×10⁷ copies/?g DNA,SHIV content were markedly different in different Litopenaeus vannamei.In order to further study the susceptibility tissues and organs of the SHIV,we carry out the SHIV artificial infection experiment to Litopenaeus vannamei by feeding with the tissue of SHIV positive shrimp.The SHIV content in the haemolymph,antennal flagellum,rostrum,gills,hepatopancreas,pleopods,muscle,and uropods were tested by real time PCR.Results showed all of the tiddues were SHIV-positive,haemolymph contained 1.20×10⁹copies SHIV/?g DNA,which was the highest concentration of SHIV copies in the tissues tested.Antennal flagellum,rostrum and uropods tissues had a mean of 2.16×08,1.34×10⁸ and 1.24×10⁸ copies SHIV/?g DNA,respectively.Pleopods,gills and hepatopancreas have lower concentration of SHIV copies,were 6.80×10⁷?3.06×10⁷? 1.52×10⁷copies/?g DNA.And,muscle contained lowest concentration of SHIV copies,which was 7.56×10⁶ copies SHIV/?g DNA on average.The SHIV content was highest in haemolymph and lowest in muscle in this study,which is consistent with WSSV content in experimentally infected Litopenaeus vannamei.The SHIV content in muscle was 1000 times lower than in heamolymph,but the WSSV content in muscle was just 10 times lower than in heamolymph.The difference showed that hemolymph cell was the target tissue of SHIV to some degree.The Taq Man real-time fluorescent quantitative PCR method established in this study showed strong specificity and high sensitivity?four copies SHIV/u L DNA?for SHIV,can be applied to detect clinical shrimps samples and shrimps samples in artificial infection experiment,providing a powerful technical means for SHIV early diagnosis and epidemiological investigation and affording a technique reference to the shrimps biosecurity system construction.At the same time,we carried on a preliminary exploration on the SHIV organization distribution in Litopenaeus vannamei using SHIV artificial infection and the established real-time PCR for SHIV in our study,to provide the basis for the collection of SHIV infected shrimps samples and provide reference for SHIV in-depth study.