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. canceri. From 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' mahidol.ac.th) to obtain a free positive control plasmid (pGEM-EHPSWP).
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.
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' biotec.or.th. To join the mailing list, please visit the AHPND Detection Google Group.
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:
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.
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.