Quantification of Enterocytozoon hepatopenaei (EHP) in Penaeid Shrimps from Southeast Asia and Latin America Using TaqMan Probe-Based Quantitative PCR

Piamsomboon, Patharapol; Choi, Seong Kyoon; Hanggono, Bambang; Nuraini, Yani Lestari; Wati, Fatma; Tang, Kathy F.J.; Park, Song; Kwak, Dongmi; Rhee, Man Hee; Han, Jee Eun; Kim, Ji Hyung

doi:10.3390/pathogens8040233

Cited by 21 publications

(13 citation statements)

References 15 publications

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“…Therefore, it is impossible to obtain accurate information regarding E. artemiae infection via microscopic examination. Based on this, highly sensitive molecular methods, such as PCR (Tang et al, 2015), nested PCR (Jaroenlak et al, 2016), qPCR (Liu et al, 2018;Piamsomboon et al, 2019), loopmediated isothermal amplification (Suebsing et al, 2013;Cai et al, 2018), and recombinase polymerase amplification (Ma et al, 2021), are used instead of microscopic methods. Compared with conventional PCR, qPCR has the advantages of high sensitivity, good repeatability and quantification.…”

Section: Discussionmentioning

confidence: 99%

“…Researchers have attempted to use qPCR to detect different microsporidia genes, such as those encoding the SSU rRNA, the polar tube protein, tubulin, and the spore wall protein (Jaroenlak et al, 2016;Han et al, 2018;Piamsomboon et al, 2019;Wang et al, 2020). Of these, the SSU rRNA gene carries the most commonly used detection target sequence.…”

Section: Discussionmentioning

confidence: 99%

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Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

et al. 2021

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Enterocytospora artemiae (EAM) mainly parasitizes the hepatopancreas of Palaemonetes sinensis. Serious infection leads to hepatopancreatic lesions, which greatly reduce the vitality of P. sinensis. Currently, EAM is detected via conventional PCR methods. However, conventional PCR has low sensitivity and cannot be used for accurate quantitative detection of EAM or its parasitic activity in host tissues. In this study, we designed a pair of specific primers based on the sequence of the ribosomal protein S9 gene (RPS9; GenBank accession number: MZ420734) to establish and optimize a SYBR Green I real-time fluorescent quantitative PCR detection method for EAM. Only EAM appeared as a bright and single target band, whereas other microorganisms did not, indicating that the primer for RPS9 had high specificity. This method displayed optimum amplification effects at an annealing temperature of 55°C, and the melting curve of the product produced a single peak. The established method showed a good linear relationship from 2.2 × 108 to 2.2 × 101 copies/μL. The relationship between the number of cycle thresholds (Ct) and the logarithm of the initial template amount (x) conformed to Ct = −3.281 log x + 36.543 (R2 = 0.998). Amplification efficiency was 101.737%, and the lower limit of detection sensitivity was 2.2 × 101 copies/μL. Good intra- and inter-group repeatability was observed within the linear range. The sensitivity of this method was more than 200 times higher than that of nested PCR. Thus, detection data obtained using this method may be useful as a technical reference for rapid and accurate identification of EAM infection and for the prevention and control of EAM during P. sinensis breeding.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

et al. 2021

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“…Enterocytozoon hepatopenaei (EHP), vermiform bodies resembling protozoan gregarines, and certain culturable Vibrio species, such as V. parahaemolyticus , V. fluvialis , V. mimicus , V. alginolyticus , and Vibrio sp. were reported as potential causative agents of the disease 3 , 6 , 8 , 9 . Furthermore, deteriorated water quality with oxygen concentrations below 3.0 mg L −1 and alkalinity below 80 ppm caused peak mortality rates during WFD outbreaks 10 .…”

Section: Introductionmentioning

confidence: 99%

Structure and co-occurrence patterns of bacterial communities associated with white faeces disease outbreaks in Pacific white-leg shrimp Penaeus vannamei aquaculture

Alfiansah

Peters

Harder

et al. 2020

Sci Rep

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Bacterial diseases cause production failures in shrimp aquacultures. to understand environmental conditions and bacterial community dynamics contributing to white faeces disease (WFD) events, we analysed water quality and compared bacterial communities in water as well as in intestines and faeces of healthy and diseased shrimps, respectively, via 16S rRNA gene sequencing and qPCR of transmembrane regulatory protein (toxR), thermolabile haemolysin (tlh), and thermostable direct haemolysin genes of pathogenic Vibrio parahaemolyticus as a proxy for virulence. WfD occurred when pH decreased to 7.71-7.84, and Alteromonas, Pseudoalteromonas and Vibrio dominated the aquatic bacterial communities. the disease severity further correlated with increased proportions of Alteromonas, Photobacterium, Pseudoalteromonas and Vibrio in shrimp faeces. these opportunistic pathogenic bacteria constituted up to 60% and 80% of the sequences in samples from the early and advances stages of the disease outbreak, respectively, and exhibited a high degree of co-occurrence. Furthermore, toxR and tlh were detected in water at the disease event only. Notably, bacterial community resilience in water occurred when pH was adjusted to 8. Then WFD ceased without a mortality event. In conclusion, pH was a reliable indicator of the WFD outbreak risk. Dissolved oxygen and compositions of water and intestinal bacteria may also serve as indicators for better prevention of WfD events. Bacterial diseases are a major problem for Penaeus vannamei pond aquaculture in Asia and Latin America. They have been causing severe annual economic losses reaching approximately USD 1 billion over last decade 1,2. Among reported bacterial diseases, acute hepatopancreatic necrosis disease (AHPND) and white faeces disease (WFD) are the most infectious and lethal ones 3,4. The latter has frequently been occurring in Asian shrimp aquaculture since 2009 3,5,6 , which reduced shrimp survival to 20-30% 6. WFD events are characterized by the presence of white faecal strings which float in the rearing water 3,6. They usually occur after approximately 50 days of culture 6 , resulting in retarded shrimp growth, unprofitable harvests, and even mass mortality 7. Loss of microvilli and subsequent lysis in hepatopancreas and midgut associated with

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“…Subsequently, Tang et al (2017) reported an EHP-like pathogen in P. vannamei farmed in Venezuela; although reported as such within their paper, the authors described low similarity in several key genes between the Latin American isolate and the type species from Thailand, sequenced by Boayke et al (2017). Further, Piamsomboon et al (2019) reported EHP infection in P. vannamei exhibiting slow growth in farms in Southeast Asia (Thailand, Vietnam and Indonesia) and Latin America (Venezuela) and contaminated Artemia using a newly designed quantitative real-time PCR (qPCR) assay based on the ß-tubulin gene of the microsporidian.…”

Section: Slow Growth and Hepatic Microsporidian Infectionmentioning

confidence: 99%

The rise of the syndrome – sub‐optimal growth disorders in farmed shrimp

Rajendran

Stentiford

Bass

2021

Reviews in Aquaculture

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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 growthaffected 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, multidimensional aetiological consideration of syndromic and emerging diseases in shrimp aquaculture.

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Quantification of Enterocytozoon hepatopenaei (EHP) in Penaeid Shrimps from Southeast Asia and Latin America Using TaqMan Probe-Based Quantitative PCR

Cited by 21 publications

References 15 publications

Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

Structure and co-occurrence patterns of bacterial communities associated with white faeces disease outbreaks in Pacific white-leg shrimp Penaeus vannamei aquaculture

The rise of the syndrome – sub‐optimal growth disorders in farmed shrimp

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