Development Of A PCR Assay For The Detection Of Enterocytozoon Hepatopenaei And The Study Of Its Infection To Litopenaeus Vannamei

Enterocytozoon hepatopenaei(EHP)is an obligate intracellular parasite that mainly resides in the hepatic tubular epithelial cells of the shrimp hepatopancreas.The mature spores are mostly oval,about 0.9 μm×1.8 μm in size,belonging to the family Microsporidia and Enterocytozoon.EHP was first reported in 2003 in the slow-growing Thailand farmed shrimp(Penaeus monodon)and was successfully isolated and identified as a new species of the genus Enterocytozoon in 2009.EHP has a wide range of hosts,and shrimp such as Litopenaeus vannamei,P.monodon,and Palaemon carincauda are the main targets of infection.EHP can infect shrimp with Hepatopancreatic microsporidiasis(HPM).Shrimp infected with EHP had no obvious pathological characteristics,mainly manifested as slow growth rate or even stopped growing,and the individual size difference was obvious.It does not cause shrimp death,but when the infection is severe,it is often accompanied by various shrimp diseases.EHP has caused great economic losses to the shrimp aquaculture industry,and has now become one of the important pathogens affecting the global shrimp aquaculture production.Since 2013,shrimp breeding bases in Liaoning,Tianjin,Jiangsu,Zhejiang,Guangdong and other provinces and cities in China have been found to be infected with EHP.The positive rate of EHP in some areas is even as high as 70% and 80%.With the trend of increasing year by year,EHP and the infectious diseases caused by it have seriously endangered shrimp farming industry in our country.In order to study the EHP infection to L.vannamei in detail and detect EHP more accurately and quickly,a more sensitive and specific EHP PCR detection method was established in this paper,and the infection of EHP on different tissues and organs of L.vannamei was studied.The experimental results are as follows:1.A new ordinary PCR detection method for EHP was established.The expected fragment is 358 bp.Compared with the previous ordinary PCR methods(EHP 510 and EHP 413),EHP 358 has higher sensitivity and better specificity.It can detect EHP DNA with a minimum of 20 copies,and does not cross-react with other common shrimp pathogens,so it is suitable for the detection of a large number of samples.2.To investigate the prevalence of EHP in Shandong,samples of L.vannamei at different times,sizes and developmental stages were collected from different areas in Shandong Province,and the EHP routine PCR detection method established in this study was used for detection.The results showed that the positive rate of EHP in 639 L.vannamei samples from four regions in Shandong province was 51.2%,and the positive rate of samples in some areas,such as Yantai Zhifu District and Binzhou Beihai New District,was even as high as 70% or more,indicating that the spread and infection of EHP in Shandong is more serious.3.The infection of EHP on different organs of L.vannamei was studied by artificial feeding.It is proved that within a short period of time(10 days),EHP can infect L.vannamei,and then expand to other different tissues and organs in the shrimp body,and the infection of different tissues and organs is different,among which the midgut is the most serious,and the average EHP load is the highest.The results of this experiment show that L.vannamei is easily infected by EHP through feeding,and EHP has a greater risk of horizontal transmission,and precautions should be strengthened in shrimp aquaculture production.