Sustainable aquaculture and aquatic resources management

A new and improved PCR detection method for Enterocytozoon hepatopenaei (EHP) based on a gene encoding a spore wall protein

Author(s): Itsathitphaisarn, O, Jaroenlak, P., Sanguanrut, P., Salachan, P.V., Wiredu-Boakye, D., Williams, B.A.P, Stantiford, G.D., Flegel, T.W., Sritunyalucksana, K. | Published: 3/7/2016 | 3519 views

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is a newly emerging disease of cultivated shrimp in Asia. Current evidence indicates that it can be associated with severe growth retardation that may not be clearly evident until the second month of culture and that it may even cause low continuous mortality in the case of very severe infections. 

Here we present a new method for detecting Enterocytozoon hepatopenaei (EHP) that has superior specificity to the first generation SSU-PCR developed in 2009 when the genetic information of EHP was still limited. Due to the urgency in stemming losses to HPM, we have decided to release this method for free, non-commercial use to the global shrimp farming community.   

The second generation EHP detection method presented here is based on a gene encoding a spore wall protein (SWP) of EHP (SWP-PCR). Results from our laboratory work revealed, in contrast to SSU-PCR, that the SWP-PCR method did not give cross reactions with DNA from crabs infected with H. eriocheir and E. canceriFrom these results, we recommend that the new SWP-PCR method replace the first generation SSU-PCR method.

The sequences of the primers for the SWP-PCR method (nested PCR) are given below and can be used freely for non-commercial applications to detecting EHP. Please contact Centex Shrimp (ornchuma.its 'at' to obtain a free positive control plasmid (pGEM-EHPSWP).

Hepatopancreatic microsporidiosis caused by Enterocytozoon hepatopenaei (EHP)

Published: 25/10/2015 | 2705 views

This disease card describes the diagnosis and range of a microsporidian shrimp pathogen, Enterocytozoon hepatopenaei (EHP), first discovered in Penaeus monodon in Thailand in 2004. It infects only the tubule epithelial cells of the hepatopancreatic tissue of shrimp.

EHP was later found to also infect P. vannamei cultivated in Thailand and is suspected to have been reported from P. japonicus in Australia in 2001. EHP has been reported from Vietnam and is associated with white faeces syndrome. It resembles an unnamed microsporidian reported in the hepatopancreas of P. monodon in Malaysia in 1989 and in P. japonicus in Australia in 2001. PCR positive results have also been obtained from P. vannamei cultivated in Indonesia and India, thus it is probable that EHP is endemic in the Australasian region.


A two-tube, nested PCR detection method for AHPND bacteria

Author(s): Sritunyalucksana, K., Dangtip, S., Sanguanrut, P., Sirikharin, R., Taengchaiyaphum, S., Thitamadee, S., Mavichak, R., Proespraiwong, P., Flegel, T.W. | Published: 20/2/2015 | 3279 views

This publication describes the AP4 method for the detection of AHPND-bacteria. The advantage of the AP4 method over the AP3 method is that the it has 100 times higher sensitivity. Because of its higher sensitivity, the bacterial culture enrichment step needed when using the AP3 with low levels of AHPND bacteria may be omitted. However, the AP4 method should not be considered as a replacement for the AP3 method but simply as an alternative choice for the users to choose should they need a more sensitive detection method.

The AP4 method has been tested with the same 104 bacterial isolates that were used for validating the AP3 detection method, and the results were identical, i.e., 100% specificity and sensitivity with the 104 isolates but at 100x lower template levels.

As with the previous announcements in this series, the AP4 method is provided for free use in the detection of AHPND bacteria. A positive control plasmid for the AP4 method will be sent out to those who are already on our mailing list as recipients of plasmids for our previous AP methods to detect AHPND bacteria. For those not already on our list, the plasmid will also be provided upon request to:  Dr. Kallaya Sritunyalucksana kallaya 'at' To join the mailing list, please visit the AHPND Detection Google Group.

Acute Hepatopancreatic Necrosis Disease Card (updated June 2014)

Published: 27/6/2014 | 3135 views

Acute Hepatopancreatic Necrosis Disease (AHPND) has recently been found to be caused by a pathogenic strain of Vibrio parahaemolyticus. This updated disease card provides a summary and guidance on:

  • Disease signs at the pond level.
  • Disease signs at animal level by histopathology, including slides.
  • Molecular diagnostic tools.
  • Host range.
  • Presence in Asia-Pacific.
  • Prevention and control.
  • Links to further information.

Announcement regarding free release of primers for specific detection of bacterial isolates that cause acute hepatopancreatic necrosis disease (AHPND)

Author(s): Flegel, T., Lo, C-F. | Published: 14/1/2014 | 3480 views

Efforts to control AHPND have been hampered by the lack of a specific and rapid detection method that could be used to determine the reservoirs of the causative bacterial isolates, to insure their absence in shrimp broodstock and post larvae, to monitor shrimp during cultivation and to aid research on possible control measures.

In Thailand and Taiwan since 2012, our two groups have been conducting cooperative research on possible PCR methods to detect isolates of AHPND bacteria. On 5 December 2013 we obtained the sequence comparison information that allowed us to prepare several test PCR detection methods, and we have spent the last 20 days validating them. Today we are announcing the best method we have found so far.

In Thailand, this research has been carried out through cooperation among researchers at Centex Shrimp (Mahidol-BIOTEC cooperative center) and  the Department of Public Health both at Mahidol University and theAquaculture Business Research Center, Faculty of Fisheries, Kasetsart University. The work has been supported since 2011 by contributory funds from many sources including the Agriculture Research and Development Agency, the National Research Council of Thailand, the Thai Commission for Higher Education, Mahidol University, the National Science and Technology Development Agency, the Patani Shrimp Farmers Club, the Surathani Shrimp Farmers Club, the Thai Frozen Foods Association, Charoen Pokphand Company, SyAqua Co. Ltd. and Thai Union Co. Ltd. In Taiwan, the research has also been supported from several sources including the Taiwan National Science Council, National Cheng Kung University (NCKU), National Taiwan University (NTU) and Unipresident Enterprises Corporation.

Acute Hepatopancreatic Necrosis Syndrome (AHPNS): Status Update

Published: 7/3/2013 | 3309 views

Since the Asia Pacific Emergency Regional Consultation on EMS/AHPNS held in August 2012 in Bangkok, Thailand, many news reports have been widely circulated that have led to various baseless speculations and conclusions on the true nature of AHPNS.

This disease update/advisory is issued to address the many circulating false and baseless speculations on the effects and spread of AHPNS in the region. NACA encourages shrimp farmers and producers in the region to properly consult fish health authorities and experts in their country for proper disease diagnosis, when early mortality is observed among cultured shrimps. Fish health authorities should then officially report confirmed cases of AHPNS to their respective national Competent Authority.

Acute Hepatopancreatic Necrosis Syndrome: Disease card

Published: 18/9/2012 | 3555 views
In the absence of identified biotic or abiotic cause(s) of the syndrome, this disease card provides information on signs that can be used for presumptive (pond level) and confirmative (animal level) diagnosis of acute hepatopancreatic necrosis syndrom (AHPNS) of shrimp.

Heavy mortalities during the early stages of a shrimp crop are not unusual and there are a variety of management and pathogen related factors that can cause such losses, which are often described by the catch-all term "early mortality syndrome". However, in 2010 a new and distinctive pattern of mortalities began to be noticed, affecting both Penaeus vannamei and P. monodon.

The syndrome involves mass mortalities of up to 100% during the first 20-30 days after stocking. Affected shrimp consistently show an abnormal hepatopancreas, which may be shrunken, swollen or discoloured; loose shells, corkscrew swimming, pale colouration and slow growth. Given that these symptoms appear to be distinctive, the name "acute hepatopancreatic necrosis syndrome" has been proposed as a more appropriate term, to distinguish this condition from other causes of early mortalities.

The syndrome was first reported from China and Vietnam in 2010, Malaysia in 2011. In 2012 it has also been reported in Thailand. The syndrome has caused severe economic losses throughout the region. The cause is not yet known.

This disease card was developed as a result of the Emergency Regional Consultation on Acute Hepatopancreatic Necrosis Syndrome, held in Bangkok, 9-10 August 2012. Audio recordings of the presentations made at the consultation are available for download or streaming.

Final Report of the Emergency Regional Consultation on Early Mortality Syndrome

Published: 27/8/2012 | 4840 views
On 9-10 August 2012, an emergency regional consultation on Early Mortality Syndrome (EMS) of shrimp and associated pathology described as Acute Hepatopancreatic Necrosis Syndrome (AHPNS) was held in Bangkok, Thailand. The consultation brought together over 87 participants including international shrimp health experts, national governments in the Asia Pacific region and industry stakeholders to share information on this emerging disease, its occurrence, pathology and diagnosis, and to develop a coordinated regional response to the issue.  The consultation was organised jointly by NACA and the Australian Government Department of Agriculture, Fisheries and Forestry (DAFF). Audio recordings of 19 technical presentations made at the regional Consultation meeting are available on NACA website.

Disease advisory: Infectious Myonecrosis (IMN): Status and Threat

Published: 23/12/2010 | 1763 views
Infectious myonecrosis (IMN) is a viral disease caused by infectious myonecrosis virus (IMNV). It affects Pacific white shrimp Penaeus vannamei, tiger shrimp P. monodon and blue shrimp P. stylirostris. IMN is associated with heavy losses in farmed shrimp of 40-70%.

Originally reported from Brazil, outbreaks were reported in East Java, Situbondo District in Indonesia in May 2006. In 2009 several other provinces were affected.

With the current spread of the disease there is a high threat of the disease spreading to neighbouring P. vannamei-producing countries. Suspected outbreaks should immediately be reported to the authorities. This information sheet provides more information.

Acute viral necrosis (in scallops) Disease Card

Published: 8/12/2008 | 2592 views
Acute viral necrosis (in scallops) was included in the list of QAAD reporting list for the year 2008. The disease card developed by regional resource experts from China in support of QAAD reporting system in Asia-Pacific provides information on pathogen, hosts, and confirmatory diagnostic methods. This is an emerging disease in the region and has caused significant losses to scallop culture industry in China.

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