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April 5, 2015
Recent Advances in EMS/AHPND
In the March/April 2015 issue AQUA Culture AsiaPacific, Professor Wing-Keong Ng (Universiti Sains Malaysia) writes about recent advances in the understanding and mitigation of early mortality syndrome, based on presentations at The Ninth Symposium on Diseases in Asian Aquaculture, which was held in Ho Chi Minh City, Vietnam, from November 24 to 28, 2014. The Symposium is held once every three years in different parts of the Asia-Pacific region by the Fish Health Section of the Asian Fisheries Society.
Shrimp News: What follows are excerpts from Professor Wing-Keong Ng’s report. For all the scientific details, I recommend that you get a full copy of his report (see Source below).
Professor Wing-Keong Ng Writes
The most recent disease to affect the sustainability and profitability of global shrimp aquaculture is acute hepatopancreatic necrosis disease (AHPND) or more often referred to as EMS.
It is now well known that EMS causes massive sloughing of epithelial cells of the shrimp hepatopancreas that results in early mortality usually within 30 days after stocking postlarvae.
In March 2013, through the research of Loc Tran (Nong Lam University, Vietnam) who at that time was a PhD student of Dr. Donald Lightner at the University of Arizona in the United States, the causative vector of EMS was identified as a unique strain of the bacteria Vibrio parahaemolyticus (Vp). Subsequent research conducted in the laboratories of Professor Timothy Flegel (Mahidol University, Thailand), Professor Chu-Fang Lo (National Cheng Kung University, Taiwan) and Lightner has identified a large episomal plasmid carried by Vp as the source of the EMS-causing toxins released by this unique strain of bacteria. Plasmids are small circular DNA that are physically separate from and can replicate independently of chromosomal DNA within the bacterial cell.
The EMS-causing plasmid has since been isolated and its genome sequenced. Once its genome was known, specific polymerase chain reaction (PCR) based methods were developed by Lo in cooperation with Professor Flegel to detect EMS from infected shrimp samples and the primers (strands of nucleic acid that serve as a starting point for DNA synthesis) for this were released to the public in December 2013.
The PCR detection methods based on two pairs of primers are called AHPND Primer set 1 (API) and AHPND Primer set 2 (AP2). Subsequent testing of AP2 in detecting EMS suggested 96% accuracy with occasional false positive PCR test results. In his presentation, Flegel highlighted an improved AP3 detection method (based on a toxin gene) that resulted in 100% accuracy when tested on 104 EMS-infected samples. The related primers have been made available for free since June 18, 2014, at the website of the Network of Aquaculture Centers in Asia Pacific (NACA). Using technology transferred from the University of Arizona, a commercial PCR test kit for EMS is also available.
Using PCR assay methods, Mary Maningas (University of Santo Tomas, Philippines) in her poster presentation reported the detection of EMS-causing Vp in both Penaeus vannamei and P. monodon from three farm sites in Central Luzon of the Philippines. It would seem that her study is the first to confirm the spread of EMS to the shrimp farming industry in the Philippines.
In his poster presentation, Iftikhar Ahmad (Department of Fisheries, Malaysia) reported that of the 15 confirmed (via biochemical and histopathology methods) EMS samples, the primers API, AP2 and AP3 were not able to give a positive PCR test result while the commercial detection kit (EMS-2 from IQ2000) showed 13.3% positive. The researchers reported that there may be two major groupings of Vp in Malaysia, but the reason why none of the isolates were positive against the primers API, AP2 and AP3 is currently not known.
To further complicate the interpretation of data from PCR detection methods for EMS, Professor Kwai-Lin Thong (University of Malaya, Malaysia) presented new evidence that Vp may not be the only Vibrio species capable of causing EMS. She detailed how one particular API, 2, 3 and IQ2000 PCR-positive bacteria strain was more closely related to Vibrio sinaloensis [which has been isolated from the spotted rose, Lutjanus guttatus Steindachner, in Sinaloa, Mexico] based on whole genome sequencing.
In her presentation, Dr. Indrani Karunasagar (Nitte University, India), said, “Vibrio parahaemolyticus associated with shrimp mortalities in India do not have characteristics of AHPND strains.” When interpreting polymerase chain reaction (PCR) results from EMS infected samples, she suggested that non-Vp bacteria might also show positive API, AP2 and AP3 test results since the toxin gene is located on a plasmid that is a transmissible genetic element. She further reiterated that Vp EMS-causing strains that showed positive PCR results may be non-toxigenic as the expression of the toxin gene on the plasmid is controlled by many factors. She then provided evidence from phenotypic, genotypic and genomic analysis that the shrimp disease outbreaks on the east coast of India at the end of 2013 were due to vibriosis rather than EMS.
The premise that the acquisition of new genetic material by horizontal transfer may play a pivotal role in shaping the Vp genome was further confirmed with evidence presented by Dr. Varaporn Vuddhakul (Prince of Songkla University, Thailand). She detailed how all 129 isolates of clinical and environmental Vp obtained from 2008 to 2014 in southern Thailand tested negative with AP2 and AP3, but very recent 33 isolates from five EMS infected shrimp farms located in the same area were tested positive for both primers. She reported that the DNA profiles of the Vp EMS isolates were distinct from the Vp of the clinical and environmental isolates. She postulated that the causative agent of EMS might have originated from one clone of Vp already present in the area that subsequently developed into different serotypes with a unique O antigen.
Based on these latest developments, it would seem somewhat premature at this stage to categorically identify Vp as the direct causative agent of EMS in farmed shrimp. Evidence points to a transferable plasmid that is able to encode certain toxigenic factors that cause the rapid disintegration of the shrimp hepatopancreatic structures leading to early mortality.
Professor Flegel pointed out that unlike viruses, for example the whitespot disease in shrimp, EMS bacteria couldn’t be fully controlled by elimination of carriers. He said we now know that the direct causative agents of EMS are the Pir toxins encoded from a plasmid, which are highly mobile genetic elements. Flegel recommended that closed shrimp culture systems with enhanced biosecurity protocols be implemented to address the spread of EMS in global shrimp farming systems.
Sources: 1. AQUA Culture AsiaPacific (Editor/Publisher, Zuridah Merican, email email@example.com). Recent Advances in the Understanding and Mitigation of EMS/AHPND. Wing-Keong Ng (email firstname.lastname@example.org). Volume 11, Number 2, Page 35, March/April 2015. 2. Bob Rosenberry, Shrimp News International, April 15, 2015.
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