Enterocytozoon Hepatopenaei Research Articles

Pyrococcus furiosus Argonaute Combined with Recombinase Polymerase Amplification for Rapid and Sensitive Detection of Enterocytozoon hepatopenaei

Enterocytozoon hepatopenaei (EHP) is one of the most serious pathogens in shrimp farming. This study combines recombinase polymerase amplification (RPA) with the Argonaute from Pyrococcus furiosus (PfAgo) and establishes a sensitive and reliable method for on-site detection of EHP. With careful screening of gDNA and optimization of the reaction, the method shows a good specificity and reaches a sensitivity of single copy per reaction, which is higher than the sensitivity of the currently available molecular assays. The whole procedure can be finished within 1.5 h including the sample processing time and only requires minimum laboratory support, which is user-friendly for on-site environments. This is the first application of PfAgo for the diagnosis of infectious diseases in seafood supply chains. It provides a reliable method for on-site detection of EHP in shrimp farms and establishes a groundwork for multiplex detection of important pathogens in seafood farming using PfAgo.

Analysis on the infecting ability of different concentrations of Enterocytozoon hepatopenaei (Microsporidia, Microsporea, Chytridiopsida) in Exopalaemon carinicauda (Decapoda, Caridea, Palaemonidae)

Abstract 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.

Development and Visualization Improvement for the Rapid Detection of Decapod Iridescent Virus 1 (DIV1) in Penaeus vannamei Based on an Isothermal Recombinase Polymerase Amplification Assay.

Viral diseases have seriously restricted the healthy development of aquaculture, and decapod iridescent virus 1 (DIV1) has led to heavy losses in the global shrimp aquaculture industry. Due to the lack of effective treatment, early detection and regular monitoring are the most effective ways to avoid infection with DIV1. In this study, a novel real-time quantitative recombinase polymerase amplification (qRPA) assay and its instrument-free visualization improvement were described for the rapid detection of DIV1. Optimum primer pairs, suitable reaction temperatures, and probe concentrations of a DIV1-qRPA assay were screened to determine optimal reaction conditions. Then, its ability to detect DIV1 was evaluated and compared with real-time quantitative polymerase chain reactions (qPCRs). The sensitivity tests demonstrated that the limit of detection (LOD) of the DIV1-qRPA assay was 1.0 copies μL-1. Additionally, the presentation of the detection results was improved with SYBR Green I, and the LOD of the DIV1-RPA-SYBR Green I assay was 1.0 × 103 copies μL-1. Both the DIV1-qRPA and DIV1-RPA-SYBR Green I assays could be performed at 42 °C within 20 min and without cross-reactivity with the following: white spot syndrome virus (WSSV), Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND), Enterocytozoon hepatopenaei (EHP), and infectious hypodermal and hematopoietic necrosis virus (IHHNV). In conclusion, this approach yields rapid, straightforward, and simple DIV1 diagnoses, making it potentially valuable as a reliable tool for the detection and prevention of DIV1, especially where there is a paucity of laboratory equipment.

White Feces Syndrome, A Multifactorial Syndrome of Cultured Shrimp: A Mini Review

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.

First Report of Enterocytozoon hepatopenaei Infection in Giant Freshwater Prawn (Macrobrachium rosenbergii de Man) 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.

A laboratory challenge model for evaluating enyterocytozoon hepatopenaei susceptibility in selected lines of pacific whiteleg shrimp Penaeus vannamei

Here we report for the first time a laboratory challenge model for Enterocytozoon hepatopenaei (EHP) to determine the difference of two Specific Pathogen Free (SPF) lines of Penaeus vannamei shrimp. These lines were experimentally challenged using EHP-infected fecal strings as inoculum. Real-time PCR and histopathology assays were performed to confirm EHP infection and evaluate differences in EHP susceptibility in the two genetic lines screened. Although the histopathology of the hepatopancreas tissue showed EHP lesions in both challenged groups, the histological lesions were more pronounced in one of the SPF lines. Quantitative PCR results revealed that animals displaying less hepatocellular damage have lower EHP load compared to animals displaying more pronounced pathological changes. There was no significant difference in final survival at 36 days post-infection in these lines with survival ranging between 80 and 100%. The data showed that mortality as an endpoint metric is not a suitable parameter to determine genetic susceptibility to EHP. Instead, histopathological changes in hepatopancreas, EHP load of the same tissue, and growth retardation would be better metrics to screen EHP susceptibility in P. vannamei. The results show the feasibility of screening genetic lines of P. vannamei for EHP resistance/tolerance using fecal string as an inoculum and, assessing histopathological changes, EHP load, and weight as indicators of resistance.

A loop-mediated isothermal amplification-based microfluidic chip for triplex detection of shrimp pathogens.

Decapod iridescent virus 1 (DIV1), White spot syndrome virus (WSSV), and Enterocytozoon hepatopenaei (EHP) pose serious threats to the shrimp farming. To date, early detection remains an important way to control the occurrence and diffusion of these pathogens. Here, we developed for the first time, a loop-mediated isothermal amplification (LAMP)-based microfluidic chip detection system, which could detect DIV1, WSSV, and EHP simultaneously. The limits of detection (LoD) of the system were 10 copies/reaction for EHP and DIV1, and 102 copies/reaction for WSSV. The entire detection procedure could be completed rapidly in 40 min at 63°C with 100% specificity and had no cross-reaction with other common shrimp pathogens. This newly established method was further validated using 94 Penaeus vannamei clinical samples, which were comparable to a typical qPCR assay and revealed good stability and reproducibility. These results illustrate that this LAMP microfluidic chip detection system allows rapid triplex pathogen analysis and could satisfy the demands of the field and routine diagnoses in aquaculture.

In Silico-Based Vaccine Design Against Hepatopancreatic Microsporidiosis in Shrimp

Enterocytozoon hepatopenaei (EHP) is a spore-forming intracellular parasite that produces an economically devastating disease in farmed shrimp called hepatopancreatic microsporidiosis (HPM), known to slow the growth of hosts in aquaculture. Spore wall proteins are implicated in the recognition of host cells in microsporidia. Many parasites have been demonstrated to detect heparin, a glycosaminoglycan (GAG) molecule present on cell surfaces, as a host. The sequencing and characterization of several EHP strains started to reveal information regarding pathogen biology, pathogenicity, and spore wall proteins must bind to the shrimp cell surface to infect it, and failure to do so results in ineffective infection. Despite these devastating complications, there is still no cure or vaccine for the parasites. In this regard, an immunoinformatics method was used to generate an epitope-based vaccine against this pathogen. The immunodominant T-cell and B-cell epitopes were identified using the spore wall proteins of EHP. The final constructed vaccine sequence was developed to be immunogenic, non-allergenic as well as have better solubility. Molecular dynamics simulation revealed significant binding stability and structural compactness. Finally, using Escherichia coli K12 as a model, codon optimization yielded ideal GC content and a higher CAI value, which was then included in the cloning vector pET2+ (a). Altogether, our outcomes imply that the proposed peptide vaccine may be a good option for HPM prophylaxis. HIGHLIGHTS Hepatopancreatic microsporidiosis (HPM) is known to slow the growth of shrimp There are approved drugs or vaccines to control this disease In-silico approach and immunoinformatics has some advantage previously in fishes The design vaccine candidate would have some advantages to combat the disease for sustainable shrimp production GRAPHICAL ABSTRACT

Multiple infections of a new-type decapod hepanhamaparvovirus (DHPV) and Enterocytozoon hepatopenaei in Korea and DHPV infectivity in Penaeus vannamei

Decapod hepanhamaparvovirus (DHPV) is a viral pathogen known to cause slow growth and reduced productivity in penaeid shrimp. In this study, a new type of suspected DHPV was detected in cultured Pacific white shrimp (Penaeus vannamei) collected from 14 farms in Korea, where growth retardation was observed. DHPV was detected in 98 of 113 collected shrimp (86.7%), and 26 of 30 collected pond water (86.7%). Overall, DHPV was detected in 14 farms (100%), and coinfection of DHPV and Enterocytozoon hepatopenaei (EHP) was observed in 10 out of 14 farms (71.4%). We sequenced the presumptive viral DNA fragment at the junction of the genes encoding the NS2 and NS1 proteins of DHPV, and the nucleotide and deduced amino acid sequences which showed less than 89% and 86% identity, respectively to those of other DHPVs available in GenBank. Similarly, comparisons of the sequenced VP region of the DHPV from P. vannamei with those of other DHPVs indicated that the nucleotide and deduced amino acid sequences showed less than 79% and 83% identity, respectively. Phylogenetic analyses based on comparisons of the two DHPV fragments indicated that the newly detected DHPV in P. vannamei can be clearly differentiated from the DHPVs currently available in GenBank. Using a laboratory bioassay, the potential risk of DHPV transmission was examined by feeding virus-infected shrimp tissue to healthy P. vannamei, and potential DHPV infection was confirmed within 7 days based on PCR analysis and histopathological examination. The emergence of a new type of DHPV and its potential coinfection with EHP would have a significant impact on the global shrimp industry, and consequently, the presence of this virus in cultured shrimp as well as in the rearing pond water should be closely monitored to control its potential spread.

A one-pot RPA-CRISPR detection method for point-of-care testing of Enterocytozoon hepatopenaei infection in shrimp

The pathogen Enterocytozoon hepatopenaei (EHP) has significantly affected the shrimp farming industry since the last decade. For effective prevention, accurate and reliable detection methods for EHP infection are important, especially the methods suitable for point-of-care testing (POCT) environment. By combining recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system, this study developed a POCT-friendly detection method for EHP infection with one-pot procedure. The combined method utilized robust amplification capacity of RPA and high specificity of CRISPR, thus had better accuracy and reliability. The one-pot procedure was achieved by careful design and screening of the crRNA and systematic optimization of the reaction. The issues related to the compatibility of RPA and Cas12a cleavage have been successfully solved. The method was highly specific and the sensitivity reached 101 gene copies per reaction. The practical performance was validated with clinical shrimp samples. With the generous temperature requirement and convenient end-point reading of results, the one-pot method is highly suitable for POCT environment. It is a good example of one-pot RPA-CRISPR detection assay for POCT purpose, and has great application value in prevention of EHP infection in shrimp farming industry.

Whole-genome sequencing and comparative genomic analysis of a pathogenic Enterocytozoon hepatopenaei strain isolated from Litopenaeus vannamei

Whole-genome sequencing and comparative genomic analysis of a pathogenic Enterocytozoon hepatopenaei strain isolated from Litopenaeus vannamei

Down-Regulation of Lipid Metabolism in the Hepatopancreas of Shrimp Litopenaeus vannamei upon Light and Heavy Infection of Enterocytozoon hepatopenaei: A Comparative Proteomic Study.

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.

A modification of nested PCR method for detection of Enterocytozoon hepatopenaei (EHP) in giant freshwater prawn Macrobrachium rosenbergii.

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 (EHP Mr ) 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 (EHP Lv ). A nested SWP-PCR method was modified to fix the bug that the original inner primers could not recognize the EHP Mr strain. The redesigned inner primers successfully amplified a product of 182 bp for both the EHP Mr strain and the EHP Lv 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.

Rapid visual detection of Enterocytozoon hepatopenaei by recombinase polymerase amplification combined with lateral flow dipstick

Enterocytozoon hepatopenaei (EHP) is a high-impact pathogen in shrimp farming, causing huge economic losses to the global shrimp farming industry every year. However, current EHP detection methods are primarily based on the development of polymerase chain reaction (PCR) techniques that rely on sophisticated and expensive instruments. Consequently, a rapid, practical, and sensitive protocol for the detection of EHP is necessary. Recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) assay was developed using a pair of primers and nfo-probe targeting the conserved region of the spore wall protein gene. Under optimized reaction conditions, the LFD-RPA assay can detect 10 copies/μL of standard plasmid within 20 min at 40°C. Furthermore, the specificity of the LFD-RPA was also verified with other common pathogens of shrimp. Thirty-nine samples of Litopenaeus vannamei were collected in shrimp farms and detected using LFD-RPA and nested PCR. Thirty-two positive samples were detected by LFD-RPA. Compared with those of nested PCR, the diagnostic sensitivity and specificity of LFD-RPA were 100% and 100%, respectively. These results indicated the great application potential of the newly developed LFD-RPA assay for point-of-care diagnosis, epidemic surveillance, and epidemiological investigation of EHP.

The emerging pathogen Enterocytozoon hepatopenaei drives a degenerative cyclic pattern in the hepatopancreas microbiome of the shrimp (Penaeus vannamei)

The microsporidian Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that causes substantial economic losses in shrimp (Penaeus spp.) aquaculture worldwide. To prevent diseases in shrimp, the manipulation of the gut microbiota has been suggested. However, prior knowledge of the host-microbiome is necessary. We assessed the modulation of the microbiome (bacteria/fungi) and its predicted functions over the course of disease progression in shrimp experimentally challenged with EHP for 30 days using high throughput 16S rRNA and ITS amplicon sequencing. Infection grade was assessed for the first time by quantitative digital histopathology. According to the infection intensity, three disease-stages (early/developmental/late) were registered. During the early-stage, EHP was not consistently detected, and a high diversity of potentially beneficial microorganisms related to nutrient assimilation were found. In the development-stage, most of the shrimp start to register a high infection intensity related to a decrease in beneficial microorganisms and an increase in opportunistic/pathogenic fungi. During late-stage, animals displayed different infection intensities, showed a displacement of beneficial microorganisms by opportunistic/pathogenic bacteria and fungi related to pathogen infection processes and depletion of energetic reserves. The degenerative cyclic pattern of EHP infection and its effects on beneficial microorganisms and beneficial functions of the shrimp hepatopancreas microbiome are discussed.

Artificial germination of Enterocytozoon hepatopenaei (EHP) spores induced by ions under the scanning electron microscope

Enterocytozoon hepatopenaei (EHP), is an emerging microsporidian pathogen responsible for hepatopancreatic microsporidiasis (HPM) in shrimps and is associated with severe growth retardation. The disease causes economic losses in shrimp aquaculture. In this study, EHP spore germination was induced and demonstrated with a scanning electron microscope (SEM). The ions (cations and anions) generated by high-energy electrons during frozen water radiolysis in the SEM specimen chamber induce EHP spore germination. This study is the first to demonstrate the induction of a microsporidian spore germination by ions generated under SEM. This study will enhance our understanding of EHP biology, life cycle and lead to the development of prophylactics and therapeutics for EHP control. Also, this method will help standardize the study of germination in other microsporidians.

Validation of qPCR from a crude extract for the rapid detection of white spot syndrome virus.

White spot disease (WSD) has posed a serious threat to the China and the global shrimp aquaculture. In order to diagnose white spot syndrome virus (WSSV) early and prevent the spread and outbreak of WSD, it is necessary to establish a highly sensitive WSSV diagnosis method suitable for shrimp farming sites. In this study, a pre-amplification qPCR assay from the crude extract of samples heated lysis was established, which was further compared with the universal qPCR assay to verify the shrimp samples. The limit of detection (LOD) of pre-amplification qPCR assay and universal qPCR assay was 2.80 copies and 20.57 copies per reaction at 95% CI, respectively. It had good WSSV specificity and did not show cross-detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreas necrosis disease (AHPND), necrotizing hepatopancreatitis bacteria (NHPB), and decapod iridescent virus 1 (DIV1). A total of 36 shrimp samples were detected as WSSV DNA positive by pre-amplification qPCR with crude extract from samples heated lysis and universal qPCR with DNA extraction. The diagnostic sensitivity and specificity were 97.22% (85.5 ~ 99.9%, 95% CI) and 100% (81.5 ~ 100%, 95% CI), respectively. The agreement Kappa value was 0.959 (0.879 ~ 1, 95% CI), and the analysis results were basically consistent. Eliminating the tedious steps of extracting DNA and using pre-amplified qPCR to detect WSSV in shrimp, it is a good choice for aquaculture farms.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10499-022-00920-9.

Applying Modified VP53A Recombinant Protein as an Anti-White Spot Syndrome Virus Biological Agent in Litopenaeus vannamei Farming.

Shrimp farming is an important economic activity. However, due to the spread of pathogens, shrimp aquaculture is becoming increasingly difficult. Many studies have confirmed that white spot syndrome virus (WSSV) recombinant proteins can inhibit viral infection. Among them, VP53 recombinant protein has been found to reduce mortality upon WSSV challenge. This study was conducted in Kaohsiung, Taiwan and reports the first field feeding trial to demonstrate that WSSV recombinant proteins can improve shrimp survival rates at a farming scale. Prior to the feeding trial, the shrimp were confirmed to be slightly infected with WSSV, Vibrio parahaemolyticus strains causing acute hepatopancreatic necrosis disease (AHPND), non-AHPND V. parahaemolyticus strains, and Enterocytozoon hepatopenaei (EHP), which are common pathogens that shrimp farmers often face. The shrimp were then divided into two groups: a control group (C group) fed with a commercial diet and a protein group (P group) fed with the same commercial feed with VP53 recombinant protein. Our findings indicated that the survival rate and expression of immune genes of the P group were higher than those of the C group. The intestinal microbiota of the two groups were also analysed. Collectively, our results confirmed that the recombinant WSSV envelope protein derivative can be used as an effective anti-virus biological agent in shrimp farms.

Propionigenium and Vibrio species identified as possible component causes of shrimp white feces syndrome (WFS) associated with the microsporidian Enterocytozoon hepatopenaei

White feces syndrome (WFS) in cultivated shrimp is characterized by white shrimp midguts (intestines) and white fecal strings that float as mats on pond surfaces. The etiology of WFS is complex, but one type called EHP-WFS is associated with the microsporidian Enterocytozoon hepatopenaei (EHP). The hepatopancreas (HP), midgut and fecal strings of EHP-WFS shrimp exhibit massive quantities of EHP spores together with mixed, unidentified bacteria. In EHP-WFS ponds, some EHP-infected shrimp show white midguts (WG) and produce white feces while other EHP-infected shrimp in the same pond show grossly normal midguts (NG) and produce no white feces. We hypothesized that comparison of the microbial flora between WG and NG shrimp would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this, we selected a Penaeus vannamei cultivation pond exhibiting severe WFS and used microscopic and microbial profiling analyses to compare WG and NG samples. Histologically, EHP was confirmed in the HP and midgut of both WG and NG shrimp, but EHP burdens were higher and EHP tissue damage was more severe in WG shrimp. Further, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium burden in the HP of WG shrimp (9364 copies/100 ng DNA) was significantly higher (P = 1.1 × 10-5) than in NG shrimp (12 copies/100 ng DNA). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as potential component causes of EHP-WFS in P. vannamei.

Establishment and application of a TaqMan probe-based qPCR for the detection of Enterocytozoon hepatopenaei in shrimp Litopenaeus vannamei.

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.