Bioassay for spore polar tube extrusion of shrimp Enterocytozoon hepatopenaei (EHP)

Aldama-Cano, Diva January; Sanguanrut, Piyachat; Munkongwongsiri, Natthinee; Ibarra‐Gámez, Jose Cuauhtémoc; Itsathitphaisarn, Ornchuma; Vanichviriyakit, Rapeepun; Flegel, Timothy W.; Sritunyalucksana, Kallaya; Thitamadee, Siripong

doi:10.1016/j.aquaculture.2018.02.039

Cited by 47 publications

(33 citation statements)

References 27 publications

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“…The pathogens of EHP are hard to eliminate. Complete inhibition of the activity of EHP spores is demonstrated either by freezing the spores at −20 °C for at least 2 h or by treating them with chemicals ( Aldama-Cano et al, 2018 ). It is difficult to distinguish the EHP infected ones with normal ones, and many molecular detection methods have been developed to detect the pathogens.…”

Section: Discussionmentioning

confidence: 99%

Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

Cai

FanDe²,

Xu³

et al. 2018

PeerJ

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BackgroundEnterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP.MethodsA rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples.ResultsPositive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP.DiscussionIn this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

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Section: Discussionmentioning

confidence: 99%

Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

Cai

FanDe²,

Xu³

et al. 2018

PeerJ

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“…To better understand the kinetics and mechanics of PT germination, we performed highspeed, live-cell imaging to capture in vitro PT germination events in three microsporidian species that infect humans: A. algerae, E. hellem, and Encephalitozoon intestinalis. Although the in vivo triggers for PT firing are not well understood, in vitro triggers have been reported [53][54][55][56]. We used small variations of these conditions to optimize PT firing in vitro for our light microscopy assay (see Methods), and captured the entire germination process, including release of the cargo after transport through the PT (Fig 4A, S3-S5 Videos, S2 Table).…”

Section: Kinetics Of Polar Tube Germinationmentioning

confidence: 99%

3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery

et al. 2020

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Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm�s-1 , and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.

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“…To better understand the kinetics and mechanics of PT germination, we performed high-speed, live-cell imaging to capture in vitro PT germination events in three microsporidian species that infect humans: A. algerae, E. hellem, and Encephalitozoon intestinalis. Although the in vivo triggers for PT firing are not well understood, in vitro triggers have been reported [46][47][48][49] . We used small variations of these conditions to optimize PT firing in vitro for our light microscopy assay (see Methods), and captured the entire germination process, including release of the cargo after transport through the PT ( Fig.…”

Section: Kinetics Of Polar Tube Germinationmentioning

confidence: 99%

3-Dimensional Organization and Dynamics of the Microsporidian Polar Tube Invasion Machinery

Jaroenlak

Cammer

Davydov

et al. 2020

Preprint

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Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process in vitro. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm⋅s -1 , and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is approximately 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.

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Bioassay for spore polar tube extrusion of shrimp Enterocytozoon hepatopenaei (EHP)

Cited by 47 publications

References 27 publications

Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery

3-Dimensional Organization and Dynamics of the Microsporidian Polar Tube Invasion Machinery

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