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Microsporidia are obligate, intracellular, spore-forming parasites, whose hosts involve almost all animals from invertebrates to vertebrates. In the last decade, the microsporidium Enterocytozoon hepatopenaei (EHP) mainly infects the hepatopancreas of shrimp to hinder the host's growth, causing substantial economic losses in farms. Polar tube, as an important taxonomy index, is a unique highly specialized invasion apparatus of microsporidia. It is composed of polar tube proteins (PTPs), and more than six polar tube proteins have been identified from various microsporidia. In this study, we have successfully identified the polar tube protein 2 of EHP. EHPPTP2, rich in lysine, encoded 284 amino acids, and it contained a signal peptide of 15 amino acids at the N-terminus. The sequence alignment analysis revealed that EHPPTP2 was homologous with other polar tube protein 2 from microsporidia Ent. bieneusi , Encephalitozoon cuniculi , Enc. hellem , Enc. intestinalis and Enc. romaleae. Additionally, EHPPTP2 was expressed in mature EHP spores. Indirect immunofluorescence analysis (IFA) and immunoelectron microscopy (IEM) results showed that EHPPTP2 was localized on the whole polar tube of germinated spores. This is the first study to identify and characterize the polar tube protein from aquatic microsporidia, and these results could serve for the following epidemiological study and provide a reliable target for EHP diagnosis. • The first report for identification and characterization polar tube protein from EHP in shrimp Litopenaeus vannamei. • EHP spores could be stimulated to germinate by KOH, and the discharged polar tube of EHP could be detected by anti-EHPPTP2 serum. • Based on the anti-EHPPTP2 serum, an evaluation method for EHP spore germination activity would be developed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Current insights into EHP life cycles and transmission methods are discussed. • Methodologies of EHP diagnosis are comprehensively reviewed. • Infection with EHP and white feces syndrome appear to be conditionally related. • Practical control strategies and urgent research needs are summarized. Disease is a major limiting factor in the global production of cultivated shrimp. The microsporidian parasite Enterocytozoon hepatopenaei (EHP) was formally characterized in 2009 as a rare infection of the black tiger shrimp Penaeus monodon. It remained relatively unstudied until mid-2010, after which infection with EHP became increasingly common in the Pacific whiteleg shrimp Penaeus vannamei , by then the most common shrimp species farmed in Asia. EHP infects the hepatopancreas of its host, causing hepatopancreatic microsporidiosis (HPM), a condition that has been associated with slow growth of the host in aquaculture settings. Unlike other infectious disease agents that have caused economic losses in global shrimp aquaculture, EHP has proven more challenging because too little is still known about its environmental reservoirs and modes of transmission during the industrial shrimp production process. This review summarizes our current knowledge of the EHP life cycle and the molecular strategies that it employs as an obligate intracellular parasite. It also provides an analysis of available and new methodologies for diagnosis since most of the current literature on EHP focuses on that topic. We summarize current knowledge of EHP infection and transmission dynamics and currently recommended, practical control measures that are being applied to limit its negative impact on shrimp cultivation. We also point out the major gaps in knowledge that urgently need to be bridged in order to improve control measures. [ABSTRACT FROM AUTHOR]

[Display omitted] • A newly developed PCR assay could detect as low as 2 × 101 copies of EHP. • The assay was specific for EHP, and had no cross reaction with five other pathogens. • The specificity and sensitivity of this assay was better than the published EHP PCR methods. • 639 P. vannamei samples from Shandong, China were 51.2% EHP positive in this assay detection. Enterocytozoon hepatopenaei (EHP), a recently reported pathogen in the penaeid shrimp, is spreading widely and seriously threatening Penaeus (Litopenaeus) vannamei aquaculture. This study aimed to develop a new and more sensitive polymerase chain reaction (PCR) method for the effective detection of EHP. An EHP PCR assay with a pair of primers specifically amplifying a 358 bp EHP DNA fragment was developed, which was demonstrated to be capable of detecting as low as 2 × 101 copies of EHP and is specific for EHP without cross reaction with DNA samples prepared from five common shrimp pathogens, including white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic virus (IHHNV), hepatopancreatic parvovirus (HPV), infectious myonecrosis virus (IMNV), and yellow head virus (YHV). This new assay is more specific and more sensitive than the previously published EHP PCR methods. With the PCR assay developed in this study, we investigated the prevalence of EHP in four areas of Shandong, China by testing a total of 639 shrimp samples collected from Yantai, Binzhou, Dongying, and Weifang. The results showed that the EHP positive rate reached 51.2%, indicating that EHP is prevalent in shrimp culture in China. [ABSTRACT FROM AUTHOR]

[Display omitted] • This LAMP is a closed tube and comparatively simple and specific EHP diagnostic assay. • This LAMP assay is developed based on EHP spore wall protein (SWP) gene sequence. • This LAMP can detect EHP in all clinical samples such as Hepatopancreas, faeces, pond water and soil. • In combination with FTA cards this assay can detect EHP within 1 h (LAMP-40 min, DNA elution – 17 min). • This EHP LAMP can be possibly performed by the farmer himself with a mini dry bath. Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment. [ABSTRACT FROM AUTHOR]

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) leads to significant growth retardation of Penaeus vannamei and is increasing the economic losses in the shrimp aquaculture. Our previous metabolomic results showed that 24 metabolites including linolenic acid (LNA) significantly decreased in EHP-infected shrimp compared with healthy groups. This study aims to evaluate whether LNA can improve the growth performance and immune status of EHP-infected shrimp. The EHP-infected shrimp was fed with dietary LNA (0, 1.2, 2.4 and 4.8 g/kg diet) for 30 days. The results showed that compared to the other groups, the body length and weight of shrimp in groups of 2.4 g LNA/kg diet were significantly increased. The copy numbers of EHP were significantly reduced in group of 1.2, 2.4 and 4.8 LNA g/kg diet compared to without LNA group. Further study showed that when shrimp were fed with 2.4 g LNA/kg diet, growth-related genes (farnesoic acid O -methyltransferase and ecdysteroid-regulated protein) were down-regulated, but juvenile hormone esterase-like carboxylesterase 1 up-regulated, suggesting that LNA could improve the growth ability of EHP-infected shrimp. More importantly, three immune genes (peritrophin-44-like protein, lysozyme, and cathepsin C) in shrimp were significantly up-regulated after fed with addition of LNA. In addition, the enzyme activity of superoxide dismutase and catalase in the hepatopancreas of P. vannamei was obviously increased after fed with different concentrations of LNA. Overall, the appropriate LNA content in the feed (2.4 g/kg diet) can improve the growth of P. vannamei and enhance its non-specific immunity and antioxidant activity. These findings indicate that LNA could be used as a potential metabolic modulator for controlling the HMP caused by EHP, and shed some light on host metabolic prevention to infectious diseases. • These findings indicate that LNA could be used as a potential metabolic modulator for controlling the HMP caused by EHP. • This study is the first to report on the treatment or improvement of HPM in shrimps. • This finding could provide important information to reduce the economic losses of shrimp production caused by HPM. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP) is a newly emerging microsporidian parasite that causes growth retardation in cultured shrimp. Currently, EHP infection can be detected by standard polymerase chain reaction (PCR)-based methods, as well as loop-mediated isothermal amplification (LAMP) methods that target EHP's small subunit (SSU) ribosomal RNA (rRNA) gene (LAMP-SSU). However, this gene target has a propensity for generating false positive test results due to cross-reactivity of LAMP primers with SSU-rRNA genes from closely related microsporidia that infect other aquatic organisms. Herein, we describe the development of a simple, specific and sensitive method for detection of EHP by the naked eye. The method is a LAMP technique targeting the spore wall protein (SWP) gene combined with DNA-functionalized gold nanoparticle probes (LAMP-SWP). It has analytical sensitivity comparable to that of nested-PCR (10 copies of plasmid DNA; 1 pg of total DNA) and other reported LAMP methods. Furthermore, no cross-reactivity was observed with other pathogens known to infect shrimp. LAMP-SWP had a sensitivity of 97% (95% CI: 88.2–99.9%) and specificity of 100% (95% CI: 88.4–100%)for EHP detection, while the corresponding sensitivity and specificity of the LAMP-SSU we used for comparison were 100% (95% CI: 91.9–100%) and 56% (95% CI: 37.4–74.5%), respectively. Hence, our novel colorimetric LAMP-SWP combines high specificity for EHP (due to the SWP target), high sensitivity for 10 target copies (similar to conventional nested-PCR), confirmation of PCR amplicons by DNA hybridization and colorimetric visualization of PCR amplicons by the naked-eye (i.e., no electrophoresis or fluorometric equipment needed). It is a rapid method that could allow for real-time management decisions at shrimp hatcheries and farms without the need for sophisticated and expensive laboratory equipment. • The first report of LAMP targeting spore wall protein (SWP) gene combined with colorimetric AuNPs assay established to detect EHP in shrimp. • The selection of SWP gene as a specific target for EHP detection offers a more satisfactory performance relative to targeting the SSU rRNA gene. • The combination of LAMP-SWP and colorimetric AuNPs assay described in this work can be used as a reliable method for identification of EHP in shrimp. • The system developed in this work is practically suitable for use especially in settings with the limited testing infrastructure. [ABSTRACT FROM AUTHOR]

A double fluorescent staining method was used to differentiate life stages of Enterocytozoon hepatopenaei (EHP) with a chitin-binding dye, calcofluor white (CFW) and a nucleic acid dye, acridine orange (AO). The unreleased spores showed a significantly blue fluorescent chitin wall with weak orange-red RNA staining, while the plasmodia appeared grape cluster-like orange-red nucleic acid-containing particles without or with very weak blue fluorescence and sporophytes showed grape cluster-like orange-red particles with varying degree of blue fluorescence. The analysis of the red, blue and green intensity of different targets revealed that the ratio of relative concentration of CFW and AO (RR C/A) of plasmodia (0.36 ± 0.15) (n = 60), sporophytes (1.25 ± 0.41) (n = 157), and unreleased spores (3.06 ± 1.09) (n = 21) had extremely significant differences (P <.01), which indicated an incremental relative amount of chitin to RNA in the three stages. The quantitative analysis proposed in this study indicated that double staining with CFW and AO can provide a method for differentiating various life stages of EHP in the infected hepatopancreatic cells. • The life stages of Enterocytozoon hepatopenaei were distinguishable and countable. • Double staining with calcofluor white and acridine orange can indicate life stages. • Mature spores reduce cellular activities and sporophytes start chitin synthesis. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = −3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming. [ABSTRACT FROM AUTHOR]

• The stain calcofluor white was used to detect Enterocytozoon hepatopenaei spores. • Spores were detected in spore suspensions, infected tissues, and feces. • Calcofluor white staining of feces is a noninvasive EHP monitoring method. A fluorescent stain, calcofluor white (CFW), was used for detection of spores of the microsporidium Enterocytozoon hepatopenaei (EHP). EHP spores in suspension, in feces, or in the infected hepatopancreas of the shrimp Penaeus vannamei , can be easily stained with this chitin and cellulose binding dye to show distinct blue-white fluorescent oval walls. The dye does not stain the host tissues. EHP spores showed orange-red spots by staining with hematoxylin and phloxine (H&P) in the section. CFW staining provides a simple and rapid method for determining the presence of EHP spores in fecal or tissue samples. [ABSTRACT FROM AUTHOR]

• Enterocytozoon hepatopenaei challenge methods were compared to produce inoculum for research. • Reverse gavage and hepatopancreas injection initiated intense EHP infection in shrimp. • HP injection could be a robust and rapid means of amplifying EHP inoculum. The microsporidium Enterocytozoon hepatopenaei (EHP) is considered as an emerging pathogen threating the shrimp industry worldwide. It is an intracellular parasite that has been associated with retarded growth syndrome and white feces syndrome in shrimp. Although the impact of EHP to the shrimp industry is well known, many aspects of host-pathogen interactions are not well understood. A major limitation in the study of EHP is the lack of a reliable method to produce large quantities of inoculum rapidly and reproducibly. The present study was designed to compare different challenge methods including intramuscular injection, oral administration, co-habitation, hepatopancreas (HP) injection and reverse gavage. The results showed that the HP injection and the reverse gavage are two promising methods to infect shrimp rapidly and generate inoculum in a reproducible manner starting with a limited amount of inoculum. Therefore, the HP injection and reverse gavage were chosen for a scale-up study. Histopathology results showed that EHP proliferated in the epithelial cells of the HP in shrimp challenged via direct injection of inoculum into HP and reverse gavage treatments. In accordance with the histopathology results, the qPCR data showed that EHP loads in the challenged shrimp increased significantly with the HP injection and reverse gavage methods. Furthermore, the histopathological and quantification results indicate that HP injection and reverse gavage are two novel methods that can be used in EHP-challenge studies and for rapidly generating viable EHP inoculum. [ABSTRACT FROM AUTHOR]

White feces syndrome (WFS) is an emerging and poorly described disease characterized by the presence of floating white fecal strings in shrimp (Penaeus monodon and P. vannamei) grow-out ponds. WFS has been associated with several pathogens, including Enterocytozoon hepatopenaei. This association is based on the fact that in areas where E. hepatopenaei has been reported, there was also a high WFS prevalence. E. hepatopenaei is an emerging pathogen that has affected cultured shrimp in Indonesia, Vietnam, China, Thailand, and India. In 2016, we reported the presence of E. hepatopenaei in farmed P. vannamei in Venezuela. In this study, we describe the first case of WFS in Venezuela associated with E. hepatopenaei. The white fecal strings and shrimp displaying white feces along the gastrointestinal tract observed in this study were similar to the gross signs found in WFS-impacted P. vannamei in SE Asian countries. Furthermore, we describe a strong association between WFS and E. hepatopenaei in the samples obtained from Venezuela and Indonesia. Quantification of E. hepatopenaei in WFS-affected ponds, ponds with a history of WFS, and ponds with no WFS showed that E. hepatopenaei loads were significantly higher in WFS-affected ponds. Furthermore, these findings constitute the first report of WFS being associated with E. hepatopenaei in farmed shrimp in Latin America. Additionally, we propose that the gross signs of WFS such as floating whitish fecal strings can be used as an indicator of the presence of E. hepatopenaei in countries where E. hepatopenaei is endemic.

We developed a qPCR assay based on the β-tubulin gene sequence for the shrimp microsporidian parasite Enterocytozoon hepatopenaei (EHP). This assay reacted with the hepatopancreas (HP) of EHP-infected shrimps, and the highest copy numbers were found in HP and feces samples from Southeast Asian countries (106–108 copies mg−1), while HP samples from Latin America, Artemia, and EHP-contaminated water showed lower amounts (101–103 copies mg−1 or mL−1 of water). No false positive was found with the normal shrimp genome, live feeds, or other parasitic diseases. This tool will facilitate the management of EHP infection in shrimp farms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]