| Enterocytozoon hepatopenaei has been detected in countries such as Vietnam,Indonesia,Malaysia and Thailand since the Enterocytozoon hepatopenaei(EHP)was discovered in the slow-growing Penaeus monodon cultured in Thailand.Infection of Penaeus vannamei,when the shrimp is infected with the pathogen,it will cause slow growth or stagnant growth of the shrimp,and sometimes symptoms such as white stool,but it does not affect the feeding and survival rate of the shrimp.We also detected EHP infection in samples from Japanese prawn,P.vannamei,and Macrobrachium rosenbergii grown in Taizhou,Zhejiang Province in 2013.The relative copy number of EHP SSU rDNA in the hepatopancreas of prawns was 10~3 copies/(ng).When the HpDNA)order or EHP load index is above 3,the growth of shrimp is significantly slower,indicating that the EHP of this load is at a higher risk level,which causes the problem of slow growth of shrimp to bring huge to the shrimp farming industry.Loss needs to be highly valued.In this paper,the epidemiology of EHP has been studied in depth,using spore purification,fluorescent staining,TaqMan qPCR,in situ hybridization and other techniques to understand more information about EHP,in order to understand the disease.The infection mechanism and prevention and control strategy provide technical basis.The following research contents were mainly included:1 Purification and counting of Enterocytozoon hepatopenaei sporesEHP was considered to be one of the main pathogens causing slow growth of shrimp.This pathogen has become a serious threat to the cultured shrimp in Asia in recent years.Spores are the key form for transmission of EHP among individuals and populations.Purification and counting methods of its spores are the basic technologies for studies on risk assessment and control of the disease.In this research,we purified EHP spores from total 1g hepatopancreas tissues with an average EHP loading at(4.7±2.2)×10~4 copies/ngDNA by differential centrifugation and density gradient centrifugation.The purified spores were observed under transmission electron microscope,and counted with phloxine staining on a blood counting chamber under light microscope.Purified spores of EHP were observed under the electron microscope as ellipsoid at size of 0.7-1.2?m.The concentration of EHP spores in the suspension of purified EHP spores was counted as 5.30×10~3/?l by blood counting chamber.The total number of purified EHP spores in the band of sucrose gradients was estimated as 1.06×10~6.TaqMan qPCR quantifies the copy number of the spore suspension to 2.53×10~3 copies/ul.2 Feasibility study on detection of Enterocytozoon hepatopenaei by fluorescent dyeThe use of fluorescent dyes for the detection of microsporidia has yielded good results in many species,but no such studies exist in EHP.In order to explore the feasibility of fluorescent dye detection of EHP,we first used PI,Sytox Green and Calcofluor White stained the purified spore particles showed strong white fluorescence under the microscope,but the host tissue fragments,viruses,bacteria,etc.were not stained.The dye was applied to the stains of feces,hepatopancreas and various tissue sections of EHP-infected shrimps,and the same white fluorescence signal appeared.The staining effect of feces and hepatopancreas was the most obvious.This result is more discriminating than HE-stained sections.In order to verify the accuracy of the staining results,EHP in situ LAMP and nested PCR were performed on the samples of P.vannamei showing white fluorescent particles,and the results were all positive.The above results prove that Calcofluor White staining can be used as a rapid detection method for EHP.3 Investigation on the Carrying Status of Enterocytozoon hepatopenaei in Shrimp Biological FeedBiological bait plays a key role in shrimp breeding and seedling stage.In order to investigate whether there is infection or carrying of EHP in the bait organism,this study collected a number of samples of biological bait from Zhejiang and Hainan from September 2018 to December 2018.Samples include:silkworm,stone worm and shrimp slices,sample nucleic acids were extracted and tested for EHP.The results showed that there were 4 weak positives in the160 silkworm samples,and the rest were negative;160 stone samples were negative;4 weak positives were found in 9 shrimp samples.In situ LAMP of EHP was performed on positive silkworm samples,and the results showed no hybridization signal.The artificial shrimp feeding samples were subjected to artificial feeding infection test,and after 20 days of continuous feeding,the results were all negative.The results indicate that biological bait may be a potential risk to spread pathogens as pathogen carriers during seedling or aquaculture,but low-or ultra-low-capacity carriers are not sufficient to transmit EHP.4 Evaluation of the efficacy of anti-control agents for Enterocytozoon hepatopenaeiAt present,the infection rate of EHP in China is still very high,and it is increasingly harmful to the shrimp breeding industry in China.There is an urgent need for drugs that can effectively prevent and kill the pathogen.Based on the previous research,this study carried out more screening experiments for pharmaceuticals,and optimized the drug encapsulation process to significantly reduce the diffusion time in water.In this study,the uniformly dyed feed was wrapped with egg white,gelatin,butter,lard,sodium alginate and edible oil,and the effect of the wrapping material was evaluated by determining the OD value of the dye in water at different time periods.The results showed that the OD value in the feed water wrapped with gelatin was the smallest after soaking for 10 min,20 min and 30 min.Therefore,this study used gelatin as a wrapping material for drug screening tests.In this study,the effects of 17 drugs were added on the basis of the previous ones.Four drugs that can reduce the EHP load,numbered T2,T4,J7 and F27,were finally detected by EHP infection model and real-time PCR.A test method for the effect of drug treatment on EHP loading was practiced. |
PDF Full Text Request