Studies On Moleculai Epidemiology And Recombinase Polymerase Amplification Assays For Rapid Detection Of Hepatospora Eriocheir In Jiangsu Province

Microspora is a kind of ancient mononuclear eukaryotic organism with wireless mitochondrial structure.It has a wide host range.At present,more than 150 genera and more than 1200 species have been found.From protozoa to mammals,it is also the pathogen of many economically valuable insects,crustaceans,fish and rodents.Microsporidia can infect many organs and tissues of crustaceans.In decapods,most microsporidia usually infect muscles and connective tissues.However,in 2007,a new micro sporidium,Hepatospora eriocheir,was discovered that only infected the tubular epithelial cells of the hepatopancreas of crustaceans.The Hepatospora eriocheir of Eriocheir sinensis is not letha1.However,studies have shown that it is related to hepatopancreatic necrosis syndrome(HPNS)of Eriocheir sinensis.When mixed with other pathogenic microorganisms that infect Eriocheir sinensis,it can aggravate the death of Eriocheir sinensis and bring huge economic losses to the crab breeding industry.The aim of this study was to understand the prevalence of Hepatospora eriocheir in Jiangsu Province and the pathogenesis of hepatopancreatic necrosis syndrome of Eriocheir sinensis.At the same time,based on Recombinase Polymerase Amplification(RPA),a rapid detection technology for Hepatospora eriocheir was developed,which could provide some technical support for rapid detection of microsporidia pathogens.The main research contents are as follows:1.Based on the specific primers in the literature and the method of PCR detection,the hepatic sporozoite infection of Hepatospora eriocheir in Jiangsu Province from 2017 to 2018 was investigated.From 2017 to 2018,the infection rate of Hepatospora eriocheir was low.In 2017,85 Eriocheir sinensis were randomly selected from five regions of Jiangsu Province:Nantong,Changzhou Qianhuang,Nanjing Lishui,Xinghua Anfeng and Yancheng.Among them,14 were infected with Hepatospora eriocheir,the positive rate was 16.5%.In 2018,97 Eriocheir sinensis were randomly selected from four regions of Jiangsu Province:Nanjing Lishui,Yangzhou Gaoyou,Wuxi Yixing and Suzhou.14 of them were infected with Hepatospora eriocheir,the positive rate was 14.4%.In addition,the investigation found that the crab ponds infected with Hepatospora eriocheir had poor culture environment.In the crab ponds,the water grass grew too densely,the water was turbid and the quality was poor,which could cause and intensify the stress reaction of river crabs,thus greatly increasing the infection rate.2.In this study,the pathological changes of hepatopancreas and the activities of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px),malondialdehyde(MDA)and glycogen in Eriocheir sinensis were detected and analyzed by routine paraffin section and biochemical assay.The results showed that the hepatopancreas of Eriocheir sinensis was pale yellow and the basement membrane shrunk slightly in the early stage of the disease.Then some hepatocytes contracted and deformed,the arrangement of cells was disordered,the boundary between cells gradually disappeared,some necrotic cells fell off into the lumen of hepatic tubules,the basement membrane continued to contract and deform some of them ruptured and disintegrated,and the hepatopancreas cells were separated from the basement membrane.Clearance appeared,hepatic tubules were severely damaged,the whole hepatopancreas was pasty,and atrophied to gray in the later stage.The content of total protein in necrotic hepatopancreas was significantly lower than that in normal hepatopancreas group(P<0.01).The activities of SOD,CAT and GSH-Px in necrotic hepatopancreas were decreased in varying degrees,while the content of MDA was increased to a certain extent.The activity of SOD in necrotic hepatopancreas was significantly lower than that in normal hepatopancreas group(P<0.05),while the activity of GSH-Px and CAT in necrotic hepatopancreas was significantly lower than that in normal hepatopancreas group(P<0.01),the content of MDA in necrotic hepatopancreas was significantly higher than that in normal hepatopancreas group(P<0.01),and the content of glycogen in necrotic hepatopancreas was significantly lower than that in normal hepatopancreas group.3.Based on Basic RPA,a rapid detection method for hepatic sporozoite of crab was established.The method has low requirement for equipment and can be completed at 37℃.It has strong specificity.There are no false positives in the detection of Enterocytozoon hepatopenaei,Aeromonas hydrophila,Vibrio Parahaemolyticus and Vibrio alginolyticus.Although the sensitivity of RPA is slightly lower than that of PCR,the reaction time of RPA is shorter and the requirement for equipment is lower,so this method still has obvious advantages in basic unit and field detection.