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Hepatopancreatic microsporidiosis caused by Enterocytozoon hepatopenaei (EHP) is an emerging parasitic disease causing great economic losses in almost all shrimp-growing countries. The disease is reported to be associated with severe growth retardation, size variation, and ultimately farm productivity. This study investigated the impact of various infection routes (viz. per os, cohabitation, water-borne and soil-borne) of EHP on disease progression. Per os (oral feeding) challenged shrimp acquired infection at a faster pace and could be detected in 100% of experimental shrimps by 5 days post-infection (dpi) as compared to 15 dpi by cohabitation method. The first detection of faeces of shrimp by water-borne infection was at 7 dpi compared to 15 dpi in the case of soil-borne infection. Soil- and water-borne infections were detectable in 100% of shrimps by 15 and 30 dpi, respectively. Nested PCR revealed a prepatent period of 5 days in case of oral and cohabitation challenge and 7 and 15 days in case of acquiring infection through water and soil sediments, respectively. Thus, a faster and higher level of infection was observed by oral challenge than other methods of infection. It could be inferred that the clinical course and pathogenicity of E. hepatopenaei infection at different time points is determined by the type of exposure, relative size group of the shrimp, and the quantum of infective spores (inoculum) ingested by the shrimp. [ABSTRACT FROM AUTHOR]

As the main pathogen causing growth retardation, EHP is considered to be mainly parasitic in the hepatopancreas of shrimp. However, the intestines of shrimp infected with EHP frequently exhibit syndromes such as jejunum and white midgut. Therefore, the challenge experiment was carried out in this study to compare the differences in intestinal histology, digestion and absorption, immune defense and oxidative stress of P. vannamei between the control group and EHP infection group. Histological analysis showed that EHP infection significantly damaged the intestine of the shrimp, including intestinal villus rupture and outer membrane impairment. Concurrently, EHP infection can trigger intestinal immune response, and the expression of key immune genes like Toll, myeloid differentiation factor, anti-lipopolysaccharide factor, and Relish was significantly enhanced, while the expression of IMD and alkaline phosphatase was suppressed. Additionally, antioxidant genes manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, and nuclear factor E2-related factor 2 were up-regulated to varying extents in EHP infection group, and the contents of lipid peroxides and malondialdehyde were heavily accumulated. Moreover, the expression levels of key genes involved in nutrient absorption, transport and synthesis, such as glucose transporter 1, Na+-K+ATPase, fatty acid synthase, acetyl-CoA carboxylase, rapamycin kinase, mTOR regulation-related protein, eukaryotic translation initiation factor 4E binding protein, ribosomal protein S6 kinase, were significantly up-regulated. However, the activities of amylase, lipase, and trypsin were inhibited in EHP infection group throughout the experiment. In summary, EHP infection damaged the intestine of P. vannamei, accompanied by immune response and oxidative stress. At the same time, nutrient transport and synthesis pathways were activated, while digestive enzyme activities were inhibited, indicating that in order to maintain survival, shrimps must accelerate material transport. Unfortunately, it remains in a state of nutrient deficiency that ultimately affects growth.

Enterocytozoon hepatopenaei (EHP) is a fungal pathogen that causes hepatopancreatic microsporidiosis (HPM) in shrimp, resulting in significant economic losses for the shrimp culture industry. Although the impact of EHP on hormonal regulation in shrimp has been seldom examined, it remains a crucial area of study. In this paper, we analyzed the transcriptomic data of the hepatopancreas of EHP-infected and uninfected shrimp to explore the effect of EHP on hormonal regulation. Our results identified 157 differentially expressed genes (DEGs), of which 32 genes were upregulated and 125 were downregulated. Through gene ontology (GO) and KEGG analyses of hormone regulation-related DEGs, we identified eight genes, six of which were associated with the regulation of molting and growth, one with immunity, and one with osmotic pressure regulation. The up- and downregulated pathways were mostly related to molting and growth regulation, including EGFR tyrosine kinase inhibitor resistance, thyroid hormone signaling pathway, and steroid hormone biosynthesis. Ten genes were selected randomly for qRT-PCR, and results confirmed the findings of transcriptome sequencing. This study provides essential insights into the molecular mechanisms through which EHP affects hormonal regulation in shrimp and serves as a basis for further research to control HPM. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP) is a parasite in shrimp farming. EHP mainly parasitizes the hepatopancreas of shrimp, causing slow growth, which severely restricts the economic income of shrimp farmers. To explore the pathogenic mechanism of EHP, the host subcellular construction, molecular biological characteristics, and mitochondrial condition of Litopenaeus vannamei were identified using transmission electron microscopy (TEM), real-time qPCR, an enzyme assay, and flow cytometry. The results showed that EHP spores, approximately 1 μm in size, were located on the cytoplasm of the hepatopancreas. The number of mitochondria increased significantly, and mitochondria morphology showed a condensed state in the high-concentration EHP-infected shrimp by TEM observation. In addition, there were some changes in mitochondrial potential, but apoptosis was not significantly different in the infected shrimp. The qPCR results showed that the gene expression levels of hexokinase and pyruvate kinase related to energy metabolism were both upregulated in the diseased L. vannamei. Enzymatic activity showed hexokinase and lactate dehydrogenase were significantly increased in the shrimp infected with EHP, indicating EHP infection can increase the glycolysis process and decrease the oxidative phosphorylation process of L. vannamei. Previous transcriptomic data analysis results also support this conclusion. [ABSTRACT FROM AUTHOR]

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Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects Litopenaeus vannamei, causing severe hepatopancreatic microsporidiosis (HPM) and resulting in significant economic losses. This study utilizes a combined analysis of transcriptomics and metabolomics to unveil the dynamic molecular interactions between EHP and its host, the Pacific white shrimp, during the early and late stages of infection. The results indicate distinct immunological, detoxification, and antioxidant responses in the early and late infection phases. During early EHP infection in shrimp, immune activation coincides with suppression of genes like Ftz-F1 and SEPs, potentially aiding parasitic evasion. In contrast, late infection shows a refined immune response with phagocytosis-enhancing down-regulation of Ftz-F1 and a resurgence in SEP expression. This phase is characterized by an up-regulated detoxification and antioxidant response, likely a defense against the accumulated effects of EHP, facilitating a stable host–pathogen relationship. In the later stages of infection, most immune responses return to baseline levels, while some immune genes remain active. The glutathione antioxidant system is suppressed early on but becomes activated in the later stages. This phenomenon could facilitate the early invasion of EHP while assisting the host in mitigating oxidative damage caused by late-stage infection. Notably, there are distinctive events in polyamine metabolism. Sustained up-regulation of spermidine synthase and concurrent reduction in spermine levels suggest a potential role of polyamines in EHP development. Throughout the infection process, significant differences in genes such as ATP synthase and hexokinase highlight the continuous influence on energy metabolism pathways. Additionally, growth-related pathways involving amino acids such as tryptophan, histidine, and taurine are disrupted early on, potentially contributing to the growth inhibition observed during the initial stages of infection. In summary, these findings elucidate the dynamic interplay between the host, Litopenaeus vannamei, and the parasite, EHP, during infection. Specific phase differences in immune responses, energy metabolism, and antioxidant processes underscore the intricate relationship between the host and the parasite. The disruption of polyamine metabolism offers a novel perspective in understanding the proliferation mechanisms of EHP. These discoveries significantly advance our comprehension of the pathogenic mechanisms of EHP and its interactions with the host. [ABSTRACT FROM AUTHOR]

【Objective】The aquaporin gene EHP00_492 from Enterocytozoon hepatopenaei (EHP) was cloned and its expression characteristics in EHP mature spores was analyzed, which provided a theoretical basis for studying the function of EHP aquaporin.【Method】The complete EHP00_492 gene was cloned from Ent. hepatopenaei. The sequence characteristics of EHP00_492 was analyzed by bioinformatics methods. Recombinant expression vector pCold-TF-EHP00_492 was constructed and transformed into Escherichia coli to induced fusion protein expression for preparing the rabbit polyclonal antibody of EHP00_492. The expression and subcellular localization characteristics of EHP00_492 in EHP mature spores were analyzed by Western blot and indirect immunofluorescence assay.【Result】EHP00_492 gene is 729 bp in length, encoded 242 amino acids. EHP00_492 is rich in leucine, with a predicted molecular weight of 25 kD. It has no signal peptide, but involving six transmembrane domains, the MIP conserved domain and two aquaporin conserved NPA/G motif. In addition, EHP00_492 has a high homology with other microsporidian aquaporins, and the phylogenetic tree analysis result shows that EHP00_492 is closely related with EBI_27080 protein from Ent. bieneusi. The three-dimensional structure of EHP00_492 is highly similarity with the identified aquaporin NbAQP from Nosema bombycis and EcAQP from Enc. cuniculi by AlphaFold analysis, which is speculated that EHP00_492 is a potential aquaporin. EHP00_492 is a 21 kD-protein in the mature spores of EHP by Western blot analysis. And the subcellular localization analysis showed that EHP00_492 protein was localized at the spore wall of EHP mature spores.【Conclusion】In this study, the sequence and structure characteristics of EHP00_492 protein, as well as its phylogenetic relationship with other microsporidian aquaporins, and the expression and subcellular localization characteristics of EHP00_492 protein in EHP mature spores were preliminary clarified, which provided basis for further study on the function of EHP aquaporins.

Enterocytozoon hepatopenaei (EHP) is a parasite in shrimp farming. EHP mainly parasitizes the hepatopancreas of shrimp, causing slow growth, which severely restricts the economic income of shrimp farmers. To explore the pathogenic mechanism of EHP, the host subcellular construction, molecular biological characteristics, and mitochondrial condition of Litopenaeus vannamei were identified using transmission electron microscopy (TEM), real-time qPCR, an enzyme assay, and flow cytometry. The results showed that EHP spores, approximately 1 μm in size, were located on the cytoplasm of the hepatopancreas. The number of mitochondria increased significantly, and mitochondria morphology showed a condensed state in the high-concentration EHP-infected shrimp by TEM observation. In addition, there were some changes in mitochondrial potential, but apoptosis was not significantly different in the infected shrimp. The qPCR results showed that the gene expression levels of hexokinase and pyruvate kinase related to energy metabolism were both upregulated in the diseased L. vannamei. Enzymatic activity showed hexokinase and lactate dehydrogenase were significantly increased in the shrimp infected with EHP, indicating EHP infection can increase the glycolysis process and decrease the oxidative phosphorylation process of L. vannamei. Previous transcriptomic data analysis results also support this conclusion.

Copyright of Guangdong Agricultural Sciences is the property of South China Agricultural University, Guangdong Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The objective of this work was to investigate, for the first time, the antioxidant effect of a mixture of natural antimicrobials in an Enterocytozoon hepatopenaei (EHP) shrimp-gut model of infection and the biological mechanisms involved in their way of action. The study approach included investigations, firstly, in vitro, on shrimp-gut primary (SGP) epithelial cells and in vivo by using EHP-challenged shrimp. Our results show that exposure of EHP spores to 0.1%, 0.5%, 1%, and 2% AuraAqua (Aq) significantly reduced spore activity at all concentrations but was more pronounced after exposure to 0.5% Aq. The Aq was able to reduce EHP infection of SGP cells regardless of cells being pretreated or cocultured during infection with Aq. The survivability of SGP cells infected with EHP spores was significantly increased in both scenarios; however, a more noticeable effect was observed when the infected cells were pre-exposed to Aq. Our data show that infection of SGP cells by EHP activates the host NADPH oxidases and the release of H2O2 produced. When Aq was used during infection, a significant reduction in H2O2 was observed concomitant with a significant increase in the levels of CAT and SOD enzymes. Moreover, in the presence of 0.5% Aq, the overproduction of CAT and SOD was correlated with the inactivation of the NF-κB pathway, which, otherwise, as we show, is activated upon EHP infection of SGP cells. In a challenge test, Aq was able to significantly reduce mortality in EHP-infected shrimp and increase the levels of CAT and SOD in the gut tissue. Conclusively, these results show, for the first time, that a mixture of natural antimicrobials (Aq) can reduce the EHP-spore activity, improve the survival rates of primary gut-shrimp epithelial cells and reduce the oxidative damage caused by EHP infection. Moreover, we show that Aq was able to stop the H2O2 activation of the NF-κB pathway of Crustins, Penaeidins, and the lysozyme, and the CAT and SOD activity both in vitro and in a shrimp challenge test. [ABSTRACT FROM AUTHOR]

Aquaculture combines techniques for breeding and harvesting aquatic organisms used in shrimp production. It is important as a source of income and for generating foreign exchange in the regions where it is practiced. However, the timely detection of diseases continues to be a great challenge for aquaculture and fisheries production. In recent years, Enterocytozoon hepatopenaei (EHP) has emerged as a major pathogen of the Pacific white shrimp Penaeus vannamei in many Asian countries (Vietnam, China, Indonesia, Malaysia, Thailand, India, and Korea). In Latin America, only in Venezuela, and to date, there is no report of its presence in Mexico. It is transmitted directly from shrimp to shrimp by oral or fecal means, cannibalism, or exposure to contaminated water. Hepatopancreatic microsporidiosis (HPM) is mainly associated with stunted growth and severe infections that can cause a poor production cycle, mortality, and problems in larva-producing laboratories. This review aims to overview the main microsporidian parasites and diseases found in white shrimp, including the clinical signs, control and prevention measures for EHP infection, and the detection of HPM using different techniques. In order to offer timely detection tools, different techniques are available for the detection and study of microsporidia. Such as optical microscopy, transmission electron microscopy, and histology; however, for diagnostic purposes, molecular methods are preferred due to their sensitivity, specificity, and short-time analysis. Our review suggests that constant monitoring in shrimp hatcheries and farms is essential to avoid the entry or transference of infected organisms, affecting shrimp production and the ideal development of healthy shrimp. [ABSTRACT FROM AUTHOR]

This study explores the impact of SYNLAC Prime probiotics on the growth performance, health status, and metabolic profile of white shrimp, Penaeus vannamei. Shrimp fed with the experimental diets, including the control diet without probiotic supplementation, and the diets supplemented with SYNLAC Prime probiotics at concentrations of 105 CFU (g diet)−1 (P5) and 106 CFU (g diet)−1 (P6) for 56 days. Results indicated a significant enhancement in growth performance in probiotic-treated shrimp relative to the control group, attributed to structural improvements in the digestive tract, particularly the increased abundances of B cells in the hepatopancreas. The administration of dietary probiotics markedly reduced the severity of Enterocytozoon hepatopenaei (EHP) infection and decreased cumulative mortalities following Vibrio alginolyticus challenge. Shrimp in the P6 group exhibited significant elevations in phenoloxidase activity, respiratory burst, lysozyme activity and phagocytic activity compared to control group. Furthermore, there was an upregulation of several immune-related genes in hepatopancreas, including serine protease (SP), prophenoloxidase (proPO) I, proPO II, and penaeidin 3a. Additionally, the expression of β-1, 3-glucan binding protein and SP mRNA was significantly increased in hemocytes. Untargeted metabolomics analysis using LC-MS/MS revealed significant changes in the hepatopancreas metabolic profile, highlighting alterations in energy metabolisms pathways, such as citrate cycle and nicotinate and nicotinamide metabolism, as well as amino acid metabolisms pathways including arginine and proline metabolism, taurine and hypotaurine metabolism, and histidine metabolism. These findings underscore the potential of SYNLAC Prime probiotics in enhancing shrimp growth, immune function, and metabolic pathways, offering valuable insights for advancing health management strategies in shrimp aquaculture. • SYNLAC Prime probiotics enhance shrimp growth and improve digestive tract structure. • Probiotics lower Enterocytozoon hepatopenaei infection severity and mortality post-Vibrio alginolyticus exposure. • Metabolomics shows key metabolic changes, highlighting probiotics' role in shrimp health management. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasite of shrimp, that may cause growth retardation or even death of shrimp. However, the characteristics of the EHP genome and its evolutionary relationships with other microsporidian genomes are not well understood. In this research, the whole genome of a strain of EHP isolated from Litopenaeus vannamei was sequenced, and detailed functional annotation was performed by using different public databases and genome component analysis. The genome size was 3.03 Mb, with 15 contigs and 2283 protein-coding genes. There were 46 tRNAs and 14 rRNAs (including 6 5S rRNAs, 4 18S rRNAs, and 4 28S rRNAs) in the genome. In addition, details of EHP evolution were revealed by comparative genomic analysis, including analyses of gene family clustering, phylogenetic relationships, gene family expansion and contraction, and synteny. The results will deepen our understanding of the EHP genome and its evolutionary features. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP), a microsporidian responsible for hepatopancreatic microsporidiosis, is a major pathogen in commercial shrimp production. Among the affected species, Palaemon carinicauda (formerly Exopalaemon carinicauda) is commercially important in China and represents a potential research model for studying crustaceans. However, little information is available on its response to EHP infection. Hence, this study analyzed the transcriptome and metabolome of P. carinicauda's hepatopancreas using high-throughput sequencing and liquid chromatograph-mass spectrometry (LC-MS) to determine its response during the early stage of infection. The transcriptomic analyses identified 730 differentially expressed genes, of which those associated with EHP infection were enriched in metabolic pathways as well as detoxification and antioxidant pathways. In addition, 144 differential metabolites were identified using a combination of positive and negative ion modes in LC-MS. The Kyoto encyclopedia of genes and genomes pathway analyses further indicated that the degradation of aromatic compounds, the AMP-activated protein kinase signaling pathway and C5-branched dibasic acid metabolism were significantly enriched after EHP infection. These results could provide useful insights into the effects of EHP on shrimps during the early stages of infection and help to understand the mechanisms underlying the stunted growth of shrimps after infection. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP) causes slow growth syndrome in shrimp, resulting in huge economic losses for the global shrimp industry. Despite worldwide reports, there are no effective therapeutics for controlling EHP infections. In this study, five potential druggable targets of EHP, namely, aquaporin (AQP), cytidine triphosphate (CTP) synthase, thymidine kinase (TK), methionine aminopeptidase2 (MetAP2), and dihydrofolate reductase (DHFR), were identified via functional classification of the whole EHP proteome. The three-dimensional structures of the proteins were constructed using the artificial-intelligence-based program AlphaFold 2. Following the prediction of druggable sites, the ZINC15 and ChEMBL databases were screened against targets using docking-based virtual screening. Molecules with affinity scores ≥ 7.5 and numbers of interactions ≥ 9 were initially selected and subsequently enriched based on their ADMET properties and electrostatic complementarities. Five compounds were finally selected against each target based on their complex stabilities and binding energies. The compounds CHEMBL3703838, CHEMBL2132563, and CHEMBL133039 were selected against AQP; CHEMBL1091856, CHEMBL1162979, and CHEMBL525202 against CTP synthase; CHEMBL4078273, CHEMBL1683320, and CHEMBL3674540 against TK; CHEMBL340488, CHEMBL1966988, and ZINC000828645375 against DHFR; and CHEMBL3913373, ZINC000016682972, and CHEMBL3142997 against MetAP2.The compounds exhibited high stabilities and low binding free energies, indicating their abilities to suppress EHP infections; however, further validation is necessary for determining their efficacy. [ABSTRACT FROM AUTHOR]

White feces syndrome (WFS), a gastrointestinal disorder of cultivated penaeid shrimp, causes severe economic and production losses worldwide. Shrimp with WFS usually show a reduced feed consumption and growth rate, hepatopancreatic discoloration, and loose shells. Recently, WFS has been said to be related to an infection with Enterocytozoon hepatopenaei (EHP), a microsporidian parasite, and other opportunistic bacteria, such as the Vibrio species. Vibrio spp. may play an important role in WFS occurrence; nevertheless, Vibrio sp., as a single pathogen, cannot reproduce this syndrome under laboratory conditions. To date, no novel treatment has been found for WFS; therefore, preventive measures, such as strict biosecurity systems to eliminate or inactivate EHP spores and limit the total Vibrio number, have been suggested. This review summarizes the information regarding the association between WFS and water parameters, gut microbiomes, EHP and Vibrio, and disease control strategies. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP), the fungus-related micro-organism that infects the hepatopancreas of marine shrimp, is believed to cause growth retardation of the infected shrimp. As the hepatopancreatic cells are composed of different cell types that support the digestive system and innate defense, malfunction of the infected cells could lead to growth retardation and weakness. This study aimed to determine of the percentage of each hepatopancreatic cell type infected by EHP and its infectious levels in the normal and growth-retarded Pacific whiteleg shrimp Litopenaeus vannamei. Histological examination revealed that all the cell types examined were infected with EHP, with significantly higher (p

Simple Summary: To our knowledge, this is the first reported detection of Enterocytozoon hepatopenaei (EHP) in cultured giant freshwater prawns (Macrobrachium rosenbergii) in 2021. While the prawn farms exhibited no clinical signs of EHP or mortality, histopathological and molecular biological analyses detected EHP during disease monitoring. The prevalence of EHP in infected prawn farms ranged from 4.9% to 18.2%. This is the first case of EHP infection in giant freshwater prawns cultured in the Republic of Korea. In the Republic of Korea, Enterocytozoon hepatopenaei (EHP) was first isolated from Pacific whiteleg shrimp in April 2020; however, there are no existing reports of EHP infection in other shrimp or prawns. Here, we aimed to investigate EHP infection and its prevalence in giant freshwater prawn farms in the Republic of Korea. We tested prawns from 22 farms for EHP infection, and samples from eight farms showed positive EHP infection results in 2021. In EHP-infected prawn farms, the prevalence ranged from 4.9% to 18.2%. The prevalence of EHP infection in the Republic of Korea, derived from the prevalence in prawn farms, was estimated to be 0.8% in 2021. The proliferation of EHP was observed within the hepatopancreatic epithelial cells of prawns using H&E and Giemsa staining. Mature EHP was observed in the sinus between epithelial cells of the digestive tubules. Phylogenetic analysis revealed a clade distinct from the previously reported EHP in Pacific whiteleg shrimps. This is the first report of EHP infection in a giant freshwater prawn in the Republic of Korea, where the prevalence of EHP infection is not high, but it is recognized as an emerging disease that requires periodic monitoring and quarantine management in giant freshwater prawns. [ABSTRACT FROM AUTHOR]

Exploring the relationship between the number of Enterocytozoon hepatopenaei (EHP) in the ambient water and its infecting ability in Exopalaemon carinicauda , we set up 6 EHP-concentration groups of 0, 101, 102, 103, 104, and 105. Subsequently, we compared the differentiation of the number of EHP copies in different tissues, the change in hepatopancreas structure, and the inducible nitric oxide synthase and NO in the haemolymph. Our result showed: the number of EHP copies in each tissue would increase significantly following the increase of EHP concentration in the water. The highest EHP copy numbers were found in hepatopancreas at the same infection condition. Additionally, EHP copy numbers in hepatopancreas were positively correlated with infection time. Furthermore, histopathological results showed the damage to the hepatopancreas had severed with the concentration of EHP increasing. Also, iNOS activity and NO content in the haemolymph of shrimp were affected by the different EHP concentrations. This study revealed significant differences in the infection ability of different concentrations of EHP spores. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP) is the pathogen of hepatopancreatic microsporidiosis (HPM) in shrimp. The diseased shrimp Litopenaeus vannamei exhibits a slow growth syndrome, which causes severe economic losses. Herein, 4D label-free quantitative proteomics was employed to analyze the hepatopancreas of L. vannamei with a light (EHPptp2 < 103 copies/50 ng hpDNA, L group) and heavy (EHPptp2 > 104 copies/50 ng hpDNA, H group) load of EHP to better understand the pathogenesis of HPM. Exactly 786 (L group) and 1056 (H group) differentially expressed proteins (DEPs) versus the EHP-free (C group) control were mainly clustered to lipid metabolism, amino acid metabolism, and energy production processing. Compared with the L group, the H group exhibited down-regulation significantly in lipid metabolism, especially in the elongation and degradation of fatty acid, biosynthesis of unsaturated fatty acid, metabolism of α-linolenic acid, sphingolipid, and glycerolipid, as well as juvenile hormone (JH) degradation. Expression pattern analysis showed that the degree of infection was positively correlated with metabolic change. About 479 EHP proteins were detected in infected shrimps, including 95 predicted transporters. These findings suggest that EHP infection induced the consumption of storage lipids and the entire down-regulation of lipid metabolism and the coupling energy production, in addition to the hormone metabolism disorder. These were ultimately responsible for the stunted growth. [ABSTRACT FROM AUTHOR]

The microsporidian Enterocytozoon hepatopenaei (EHP) has become a critical threat to the global shrimp aquaculture industry, thus necessitating early detection by screening. Development of a rapid and accurate assay is crucial both for the active surveillance and for the assessment of shrimp with EHP infection. In the present study, a distinct strain of E. hepatopenaei (EHPMr) was found in Macrobrachium rosenbergii. The SWP1 gene analysis revealed it was a new genotype that differed with the common strain isolated from the Litopenaeus vannamei (EHPLv). A nested SWP-PCR method was modified to fix the bug that the original inner primers could not recognize the EHPMr strain. The redesigned inner primers successfully amplified a product of 182 bp for both the EHPMr strain and the EHPLv strain. The new primers also had good specificity and high sensitivity, which may serve as an alternative for EHP genotyping. This study provided a method for detection of EHP in the biosecurity of Macrobrachium rosenbergii farming, and the developed protocol was proposed for the routine investigation and potential carrier screening, especially for molecular epidemiology. [ABSTRACT FROM AUTHOR]

Hepatopancreatic microsporidiosis (HPM), caused by the microsporidium Ecytonucleospora hepatopenaei (EHP) leads to retarded growth and enhanced susceptibility to other diseases in shrimp resulting in a major loss for the shrimp industry worldwide. It is little understood how EHP infects its host and hijacks its cellular machinery to replicate and exert clinical manifestations in infected shrimp. Since the initial record of HPM, histopathology and polymerase chain reaction (PCR)-based assays were developed for the detection of EHP to prevent spread of the disease. Availability of an antibody-based detection method would complement these existing diagnostic tools and be useful in studying EHP pathogenesis. We describe here an immunofluorescence assay (IFA) for detecting EHP using monoclonal antibodies (mAbs) that were originally developed against Cryptosporidium parvum , a coccidian parasite that infects calves (Bos taurus), other agriculturally important animals, and humans. Forty-one mAbs were screened and two mAbs, 3E2 and 3A12, were found to detect EHP successfully. The utility of these mAbs in detecting EHP was further assessed by testing 36 experimentally challenged EHP-infected shrimp (Penaeus vannamei). EHP-detection data from infected shrimp were compared by Hematoxylin and Eosin (H&E) histology, real-time PCR, and immunofluorescence. The data show IFA using mAbs 3E2 and 3A12 could successfully detect EHP and that the sensitivity of detection is comparable to H&E histology and quantitative PCR. Availability of mAbs that can detect EHP is expected to be immensely beneficial in HPM diagnosis. Since the pathobiology of C. parvum has been so widely studied, these cross-reactive mAbs may also aid in gaining some insight into EHP pathogenesis and disease. • An immunofluorescence assay (IFA) was developed to detect EHP using mAbs against Cryptosporidium parvum. • Two out of forty one mAbs, 3E2 and 3A12, were found to detect EHP successfully. • EHP-detection by IFA was comparable to H&E histology and quantitative PCR. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP) is a common parasite that invades the epithelial cytoplasm in the hepatopancreas of shrimp Litopenaeus vannamei and results in slow growth of the host shrimps to cause significant economic loss in shrimp aquaculture. In this study, a TaqMan probe–based qPCR for quantitative detection of EHP was established. A pair of specific primers and a TaqMan probe were designed based on the sequence of cysteine desulfurase gene (NFS1) of EHP. The standard curve between cycle threshold (Ct) and the logarithmic starting quantity (SQ) of the template was determined as Ct = − 3.231 lg (SQ) + 40.638, with a correlation coefficient (R2) of 0.998 and an amplification efficiency of 103.9%. The lower limit of quantification was 1.67 × 101 copies/μL for this TaqMan probe–based qPCR and 1.67 × 103 copies/μL for the conventional PCR. The TaqMan probe–based qPCR established in the research was 100 times more sensitive than the conventional PCR method. In addition, the results of clinical sample detection indicated that the present technique was efficient in detecting EHP in the hepatopancreas, feces, water, and pond bottom mud samples. Therefore, the established TaqMan probe–based qPCR is a suitable technique for detecting EHP in both shrimp and aquatic environment samples. [ABSTRACT FROM AUTHOR]

Hepatopancreatic parvovirus (HPV) and Enterocytozoon hepatopenaei (EHP) are emerging and reemerging pathogens in shrimps. In the present study, a novel genotype of HPV concurrently infected with EHP in Penaeus vannamei in Taiwan leading to severe atrophy and damage of hepatopancreas were confirmed by histopathology, in situ hybridization, and PCR. The novel genotype of HPV exhibited 66%–69.5% sequence identities with all known HPVs and carried unique amino acid deletions and insertions in the VP gene. According to phylogenetic analysis, the Taiwan HPV isolates were classified as the genotype IV. The present study not only provided the histopathological and molecular proof of HPV and EHP co‐infection in Taiwan, but also revealed the importance of investigating the geographical expansion of novel HPV genotypes. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP) and Vibrio parahaemolyticus acute hepatopancreatic necrosis disease (VPAHPND) are two of the diseases that have frequently infected farmed shrimp in recent years, causing great economic losses to the shrimp industry worldwide. In this study, we established a sensitive and accurate duplex droplet digital PCR (ddPCR) method that can simultaneously detect and quantify the two pathogens simultaneously. The results showed that the ddPCR methods could detect EHP and VPAHPND specifically. The sensitivity levels of ddPCR for EHP and VPAHPND were 2.3 copies/μl and 4.6 copies/μl, respectively, which were 10‐fold higher than the sensitivity of the qPCR assay and showed good reproducibility. Twenty‐six suspected diseased shrimp samples were used for practical determination. For EHP, the detection rates of ddPCR and qPCR were 53.84% and 42.31%, respectively; for VPAHPND, the detection rates of ddPCR and qPCR were both 23.08%. The results indicated that the ddPCR method shows superiority for detection in samples with low viral loads, which will facilitate monitoring of the source and transmission of EHP and VPAHPND and will help control shrimp epidemic disease. [ABSTRACT FROM AUTHOR]

Abstract Background Enterocytozoon hepatopenaei (EHP) is an enteric pathogen that affects Penaeus vannamei and Penaeus monodon shrimp in many SE Asian countries. In the western hemisphere, EHP was reported for the first time in 2016 in farmed P. vannamei in Venezuela. Anecdotal evidence suggests that EHP is more prevalent in grow-out ponds where the salinity is high (> 15 parts per thousand (ppt)) compared to grow-out ponds with low salinities (

Enterocytozoon hepatopenaei (EHP), the fungus-related micro-organism that infects the hepatopancreas of marine shrimp, is believed to cause growth retardation of the infected shrimp. As the hepatopancreatic cells are composed of different cell types that support the digestive system and innate defense, malfunction of the infected cells could lead to growth retardation and weakness. This study aimed to determine of the percentage of each hepatopancreatic cell type infected by EHP and its infectious levels in the normal and growth-retarded Pacific whiteleg shrimp Litopenaeus vannamei. Histological examination revealed that all the cell types examined were infected with EHP, with significantly higher (p<0.01) percentage on the M- and Fcells, compared to other cell types. The R-, B- and F-cells of the growth-retarded shrimp were infected at significantly lower (p<0.05) percentage than the same cell types of the normal shrimp. By PCR, positive reactions of EHP infection were detected in all shrimp samples, both in the normal and growth-retarded shrimp, but only at the nested PCR level. Also, the relative density of the EHP load in the hepatopancreas of the two shrimp groups did not differ statistically. These results suggest that growth retardation in L. vannamei currently exists may not be due to EHP infection. [ABSTRACT FROM AUTHOR]

Diseases in shrimp farms in the Mekong Delta of Vietnam cause significant crop losses and are therefore of great concern to producers. Once a pond becomes infected, it is difficult to prevent spread of the disease to nearby shrimp farming areas. Thus, predicting the occurrence of disease is an essential part of reducing the risk for shrimp farmers. In this study, we applied an integrated geographic information system and machine learning system to predict three serious diseases of shrimp, namely, acute hepatopancreatic necrosis, white spot syndrome disease, and Enterocytozoon hepatopenaei infection, based on data collected from shrimp farms in the Tra Vinh, Bac Lieu, Soc Trang, and Ca Mau provinces of Vietnam. We first constructed a map showing the distribution of these diseases using the locations of affected farms, and then we conducted spatial analysis to acquire the geographical features of the affected locations. This latter information was combined with environmental factors and clinical signs to form the set of independent variables affecting the outbreak of diseases. The neural network model outperformed the logistic regression, random forest, and gradient boosting methods in terms of predicting infection to estimate the probability of disease occurrence in farmed areas. Acute hepatopancreatic necrosis disease infected farms downstream of the Co Chien and Hau Rivers of Tra Vinh and west of Ca Mau. Enterocytozoon hepatopenaei infection is distributed in Soc Trang Province, while white spot syndrome virus has spread to the coastal districts of Soc Trang and Bac Lieu Provinces, where it is highly associated to water from a complex canal system. [ABSTRACT FROM AUTHOR]

Increasing evidences have showed that a tight link among pathogen infection, physiological indicators and intestinal microbiota. To investigate effects of Enterocytozoon hepatopenaei (EHP) infection to growth retardation and intestinal microbiota, different sized Pacific white shrimp (Penaeus vannamei) infecting with EHP were studied. The shrimp with big, medium and small sized groups were from same batch of postlarvae and reared in one pond under the same diet and environmental conditions. Number of EHP copies revealed that the more EHP copies the smaller size of shrimp individuals. Denaturing gradient gel electrophoresis (DGGE) profiles showed intestinal bacterial patterns of small sized shrimp were similar to the middle sized shrimp, rather than the big sized shrimp. It implied that intestinal microbiota were influenced by severity of EHP infection and suggested the different sizes of shrimp were due to the successive infection period of EHP. Among the different sized groups, the relative abundance of Vibrio of small group was the highest, suggesting EHP infection could result in higher susceptibility to pathogen invasion. Our findings provided the evidence intestinal bacterial patterns altered by infection of EHP, the more EHP copies the smaller size of shrimp, and different sized individuals due to successive infection period of EHP.

Shrimp is a globally popular seafood. Shrimp farming has been challenged by various infectious diseases that lead to significant economic losses. The prevention of two important shrimp infectious diseases, the acute hepatopancreatic necrosis disease (AHPND) and the Enterocytozoon hepatopenaei (EHP) infection, is highly dependent on early and accurate diagnostic. On‐site monitoring of the two diseases in shrimp farming facilities demands point‐of‐care‐testing (POCT) type of diagnostic assays. This study established a duplex recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) combined assay that could simultaneously diagnose the two diseases. The optimized RPA‐LFD assay could finish the diagnostic in 35 min with good specificity, and the sensitivity reached 101 and 102 gene copies per reaction for EHP and AHPND, respectively, which were at the same level as the currently available molecular diagnostic assays. Test results of clinical samples showed 100% agreement of this assay with the industrial standard nested polymerase chain reaction (PCR) assays, and samples with both diseases were simultaneously identified. Because of the isothermal 37℃ amplification and the visual reading of the signal on dipsticks, the dependence on equipment is minimal. This duplex RPA‐LFD assay is well suited for simultaneous POCT diagnostic of the two important shrimp infectious diseases. Moreover, the principle can be applied to multiplex POCT diagnostic of other infectious diseases in aquaculture. [ABSTRACT FROM AUTHOR]

Diseases caused by infectious pathogens are common in grow‐out shrimp culture. The emergence of two shrimp diseases viz. infectious myonecrosis virus (IMNV) and hepatopancreatic microsporidiosis which is caused by Enterocytozoon hepatopenaei (EHP), has resulted in substantial economic losses to shrimp farmers in India. Based on previous reports of IMNV disease outbreaks in a small cluster of farms, an attempt was made to study the horizontal spread of this disease along the east coast of India, by active surveillance in four districts of Andhra Pradesh and Tamil Nadu. The present investigation reports the cases of co‐infection of IMNV and EHP in Penaeus vannamei shrimp farms in India. Among the 12 ponds investigated, shrimp in four ponds were infected only with IMNV; a single pond was infected with EHP and seven ponds with shrimp harbouring both the pathogens. Shrimp affected by IMNV exhibited white discolouration on the abdominal segments. The EHP‐positive pond did not exhibit any apparent clinical signs. The co‐infected shrimp exhibited clinical lesions of both IMNV and EHP. Histopathology revealed that the hepatopancreas of shrimp with signs of slow growth, showed cytoplasmic developmental stages of EHP in the tubular epithelial cells and sloughing of infected cells, releasing spores in the tubular lumen whereas, IMNV affected shrimps had shredding of muscle fibres, coagulative necrosis with haemocytic infiltration in striated muscles and hypertrophy of lymphoid organ (LO). Both the infections were further confirmed by PCR as well as sequencing. [ABSTRACT FROM AUTHOR]

The phenomenon of slow growth in farmed shrimp was observed for the first time in 1989 in Penaeus vannamei as a clinical manifestation of runt deformity syndrome caused by infectious hypodermal and hematopoietic necrosis virus. Subsequently, it was recorded in Penaeus monodon during 2001–2002. Since then, the condition described as monodon slow growth syndrome (MSGS) has been reported from many countries. Though not leading to mortality, retarded growth at the pond level results in significant economic losses. Several potential pathogens have been identified from affected shrimp; however, no confirmed causal relationship has yet been established. Initially, P. vannamei farmed alongside slow growth‐affected P. monodon were unaffected by the condition. Further, experimental studies suggested that P. vannamei was not susceptible. However, in recent years, increasing incidence of severe growth retardation has been reported in farmed P. vannamei. Currently, slow growth is considered as one of the most impactful production‐limiting conditions affecting shrimp farming across Asia. Similar to MSGS, many infectious agents have been detected in slow growth‐affected P. vannamei, especially the microsporidian Enterocytozoon hepatopenaei. Further, genetic and environmental factors have been suggested to play a role. However, the exact cause of slow growth remains elusive, proposing that it may be a multi‐factor syndrome. This review tracks the trajectory of the condition over the period of significant expansion of shrimp farming and postulates that conventional single pathogen/single disease paradigms are insufficient to deal with chronic yield‐limiting syndromes. We suggest more realistic, multi‐dimensional aetiological consideration of syndromic and emerging diseases in shrimp aquaculture. [ABSTRACT FROM AUTHOR]

White feces syndrome (WFS) is a multifactorial disease that affects global shrimp production. The diagnostic approach to identify WFS involves traditional and molecular scientific methods by examining histopathology, bioassays, PCR (polymerase chain reaction), and calorimetric estimation. The pathogenesis of WFS is closely associated with Vibrio spp., intestinal microbiota (IM) dysbiosis, and Enterocytozoon hepatopenaei (EHP). It also has caused over 10–15 % loss in the aquaculture industry and is also known to cause retardation, lethargy and slowly leading to high mortality in shrimp farms. Therefore, it is necessary to understand the molecular mechanisms processed under the association of IM dysbiosis, Vibrio spp., and EHP to analyze the impact of disease on the innate immune system of shrimp. However, only very few reviews have described the molecular pathways involved in WFS. Hence, this review aims to elucidate an in-depth analysis of molecular pathways involved in the innate immune system of shrimp and their response to pathogens. The analysis and understanding of the impact of shrimp's innate immune system on WFS would help in developing treatments to prevent the spread of disease, thereby improving the economic condition of shrimp farms worldwide. [Display omitted] • White feces syndrome (WFS) causes mortality and affects global shrimp production. • WFS is mainly caused by Vibrio spp., along with other causative agents. • The innate immune system in shrimp plays the main role in the pathogenesis of WFS. • In-depth analysis of innate immune response aids in disease prevention. • This review is providing up to date comprehensive knowledge about WFS. [ABSTRACT FROM AUTHOR]

[Display omitted] • EHP infection changed the composition of gut microbiota of E. carinicauda. • Bifidobacterium animali and Lactococcus garvieae significantly reduced EHP load of EHP infecting individuals. • Bifidobacterium animali and Lactococcus garvieae improved the morphology and structure of the hepatopancreas and intestinal tissue of EHP infecting individuals. To explore the relationship between the intestinal flora of Exopalaemon Carinicauda and infection by Enterocytozoo Hepatopenaei (EHP), we analyzed the species and richness of gut microbiota in infected individuals in different EHP load groups [i.e., control (C), high load (H), and low load (L)] using gene sequencing after infection. The results showed that the abundance of intestinal flora in the high-load EHP group was significantly lower than that in the healthy group. Based on the UPGMA cluster tree and PCoA analysis, with comparisons to healthy shrimp, the gut microbiota of the EHP high load and low load groups were clustered into one branch, which indicated that EHP infection changed the composition of the gut microbiota of infected shrimps. The heat map analysis of species abundance clustering revealed that the dominant bacteria in the low EHP load group and the control group were beneficial genera such as Lactococcus , Ligilactobacillius , and Bifidobacterium , but the dominant bacteria in the high EHP load group were harmful genera such as Pseudomonas , Photobacterium, and Candidatus hepatincola. The functions of the intestinal flora predicted that most genes related to metabolism were more abundant in healthy shrimp, most genes related to metabolism and the organisms' system were more abundant in the low EHP load group, and most genes related to diseases and environmental information processing were more abundant in the high EHP load group. After separation and purification, the dominant bacteria (Bifidobacterium animalis in healthy shrimp and Lactococcus garvieae in the low EHP load group) and the non-dominant bacteria (Macrococus caseolyticus in the low EHP load group) were obtained. Each of these isolated strains were used together with EHP to infect E. carinicauda , and the results showed that Bifidobacterium animali and Lactococcus garvieae significantly reduced the EHP load in EHP-infected individuals. At the same time, the morphology and structure of the hepatopancreas and intestinal tissue of EHP-infected E. carinicauda were improved. No improvement was seen in tissue that was infected with Macrococus caseolyticus. [ABSTRACT FROM AUTHOR]

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The emerging microsporidian parasite Enterocytozoon hepatopenaei (EHP) causes retardation of shrimp growth, leading to significant financial losses in shrimp aquaculture. Therefore, the development of an efficient and sensitive detection method will be conducive to the prevention and control of the shrimp parasite. In this study, we developed and evaluated a rapid real-time recombinase polymerase amplification (RPA) method that can detect EHP within 15 min at a constant temperature of 38.5 °C. The detection limit of this EHP RPA was 10 copies/μL of DNA molecules per reaction. The specificity of EHP RPA was tested, and the assay did not cross-react with white spot syndrome virus (WSSV), shrimp hemocyte iridescent virus (SHIV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), or Vibrio parahaemolyticus. Field and clinical applicability of this assay was evaluated using 61 field samples. The coincidence rate of the detection results for the clinical samples between RPA and qPCR was 95.1 %. In summary, the real-time RPA analysis provides an efficient and sensitive detection method for EHP.

Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3–7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting ptp2 gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37 °C within 1 h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP.

Abstract Background The microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp. Results We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein. Conclusions Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.

Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3–7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms. [ABSTRACT FROM AUTHOR]

Background: Enterocytozoon hepatopenaei (EHP) is an enteric pathogen that affects Penaeus vannamei and Penaeus monodon shrimp in many SE Asian countries. In the western hemisphere, EHP was reported for the first time in 2016 in farmed P. vannamei in Venezuela. Anecdotal evidence suggests that EHP is more prevalent in grow-out ponds where the salinity is high (> 15 parts per thousand (ppt)) compared to grow-out ponds with low salinities (< 5 ppt). Considering that P. vannamei is an euryhaline species, we were interested in knowing if EHP can propagate in P. vannamei in low salinities. Results: In this study, we described an experimental infection using fecal strings as a source inoculum. Specific Pathogen Free (SPF) P. vannamei were maintained at three different salinities (2 ppt, 15 ppt, and 30 ppt) while continuously challenged using feces from known EHP-infected P. vannamei over a period of 3 weeks. The fecal strings, used as a source of EHP inocula in the challenges, was sufficient to elicit an infection in shrimp maintained at the three salinities. The infectivity of EHP in shrimp reared at 2 ppt, 15 ppt, and 30 ppt salinities was confirmed by PCR and histopathology. The prevalence and the severity of the EHP infection was higher at 30 ppt than at 2 ppt and 15 ppt. Conclusion: The data suggests that fecal strings are a reliable source of EHP inoculum to conduct experimental challenges via the fecal-oral route. An EHP infection can occur at a salinity as low as 2 ppt, however, the prevalence and the severity of the EHP infection is higher at a salinity of 30 ppt. [ABSTRACT FROM AUTHOR]

The TaqMan-MGB-based fluorescent quantitative PCR method for Enterocytozoon hepatopenaei（EHP） was developed to provide a new detection technology for quantitative detection and infection monitoring of EHP.【Method】A pair of primers and a TaqMan-Minor Groove Binder（TaqMan-MGB）probe were designed based on the conserved sequence of small subunit ribosomal DNA（SSU rDNA） gene in EHP,and the target fragments amplified by PCR were cloned into the pMD18-T vector to construct pMD18-T-SSUEHP recombinant plasmids as standards.Then the TaqMan-MGB probe fluorescent quantitative PCR method for detection of EHP was developed after optimizing primer concentration and probe concentration with standards as template,the standard curve was established for quantitative analysis with 10-time dilution of pMD18-T-SSUEHP as standards and the specificity,sensitivity,reproducibility tests and detection of clinical samples were carried out to evaluate the applicability of this method.【Result】The recombinant plasmid DNA of pMD18-T-SSUEHP had good stability and could meet the requirements of the standards and the positive control,and the standard curve showed a good linear relationship with the equation as y=-3.232×lg（x）+39.51 when quantitative range was from 2.0×101 to 2.0×109 copies/reaction.The optimized TaqMan-MGB probe fluorescent quantitative PCR method had a high sensitivity with the detection limit as low as to 20 copies/reaction for the pMD18-T-SSUEHP and approximately 10 copies/mg for the clinical shrimp samples,and its sensitivity was 10 times higher compared to the nested PCR method recommended by Ministry of Agriculture and Rural Affairs.It had no amplification for common shrimp pathogens,the variation coefficients of intra-group and inter-group were 0.26 %-0.72 % and 0.56 %-0.92 %,the entire detection could be completed within 1 h for a single sample.The TaqMan-MGB probe real-time PCR established in this study and the nested PCR method recommended by Ministry of Agriculture and Rural Affairs were applied to detect 276 clinical samples,the results showed that most of the results were the same with coincidence rate of 96.7 % and the former had better sensitivity in detecting samples with low copies EHP.The EHP positive rates of Guangxi were 16.2 % in 2018 and 30.6 % in 2019,respectively.【Conclusion】The TaqMan-MGB-based fluorescent quantitative PCR method developed in the study is rapid,sensitive,specific,reproducible and of wide quantitative range,making it an ideal method for detecting EHP in shrimp clinical samples and a new technique for the quantitative detection and infection monitoring of EHP [ABSTRACT FROM AUTHOR]