22 INFOFISH International 4/2014 www.infofish.org

Aquaculture

Early mortality syndrome – understanding the

mechanismAlthough progress has been made but the transmission mechanism of

EMS pathogens is still uncertain. The highly virulent pathogens baffling the scientists, as they genetically differ. Researchers are not getting uniform

results even though employing available sophisticated tools to save this multi-billion dollar industry. The authors have made an attempt to elucidate the

details through graphic presentation of various elements.

EMS/AHPNS is implicated in causing the 1 million mt shortage in global shrimp supply eliciting prices

to be at an all time high. The primary causative agent has been identified as a Vibrio parahaemolyticus by researchers at the University of Arizona under Prof Donald Lightner. Different strains of V parahaemolyticus can differ genetically from each other by as much as 10% while humans and chimpanzees only differ by 1.2%. Since V parahaemolyticus strains normally have 4 500 - 5 000 genes, a 10% difference means up to 500 genes could be different leading to major variations between strains.

Based on the open source AP1/AP2 PCR amplicons made public by Professor Tim Flegel and Professor Grace Lo on Christmas Day 2013 by email, we have found that the 15 V parahaemolyticus isolates tested PCR+ from EMS live shrimp samples, originating from Malaysia and Vietnam in our lab, are biochemically diverse based on API20E and API20NE biochemical screening. Pulse Field Gel Electrophoresis (PFGE) was done for DNA fingerprinting and this showed that 12 of these 15 strains were closely related to the highly virulent 3HP reference strain from CENTEX shrimp, Mahidol University. The strains we have used in challenge assays (following the method of Dr Loc Tran) vary in its speed of killing test shrimp from 1-2 days for highly virulent strains to 4-6 days for less

virulent strains. We, therefore, believe EMS is caused by a group of related strains rather than one single strain of V parahaemolyticus.

There are also 2 isolates not closely related to the reference strain that are also AP1/AP2 PCR+ that may actually be other species (V alginolyticus and V fluvialis according to API20NE assays). If so, the virulence factor which is the DNA that codes for the toxin may be able to ‘jump species’. In human disease, there are quite a number of examples where bacteriophages transfer virulence factors making non-toxic bacterial strains extremely toxic. The noteworthy ones are Cholera (CTX phage); Scarlet Fever (T12 phage); Botulism (Clostridial phages); Diphtheria (Corynephage-β) and Hemorrhagic Diarrhea (λ-phage).

In an aquaculture context, the channel catfish (Ictalurus punctatus) disease, MAS or motile Aeromonad septicaemia, bacteriophage DNA has been found

in epizootic strains of Aeromonas hydrophila that are linked to virulence associated DNA from other bacteria indicating that a lysogenic phage acted as a vehicle to historically transfer virulence genes from one bacterial species to another. We believe that this is similar to what took place in Hainan Island prior to 2009 that started off the present EMS/AHPNS contagion.

Bacteriophages are basically either lytic or lysogenic phages. Lytic ones infect bacteria, quickly multiply inside and then burst out spreading many thousands of new phages into the environment. Lysogenic ones, on the other hand, infect bacteria and integrate its genes as ‘proviral DNA’ to the bacterial chromosome such that whenever the bacteria divide, the phage DNA divides along with it. Periodically, lysogenic phages also go into a lytic cycle to release phages into the environment but sometimes these cycles may be few and far between which it is extremely difficult to isolate and photograph lysogenic phages. We believe the EMS-causing strains of V parahaemolyticus were given virulence associated DNA from another species by lysogenic phage mediated lateral transfer. Next-Gen sequencing leading to Comparative Genomics involving a broad library of EMS-causing strains compared against non-EMS wild-type strains will provide solid evidence of this as well as

by Ung Eng Huan, Thong Kwai Lin and Yew Sh Min

Thong Kwai LinUng Eng Huan

www.infofish.org INFOFISH International 4/2014 23

Aquaculture

Ung Eng Huan is currently Chief Technology Officer of Biovalence

S/B, a leading Malaysian drug discovery company. He is a co-

inventor of RetroMAD1 that kills WSSV as an oral delivery protein. He directs EMS/AHPNS research in Biovalence with Yew Sh Mynn reporting directly to him. Prof

Thong Kwai Lin, currently a Director of Research Services, IPPP, University of Malaya. Her research interests are molecular epidemiology, diagnostics

and characterisation of foodborne bacterial pathogens.

to characterise the toxin sequence itself. Based on Transmission Electron

Microscope pictures, the basement membrane cells of the hepatopancreal tubules in EMS/AHPNS shrimp form ‘blebs’ leading to cell death. Blebs are formed when the f-actin scaffolding of the basement cells are compromised possibly by a high positively charged toxic protein. Therefore, we believe this toxin to be a highly positive charged protein manufactured by the EMS-strains of V parahaemolyticus in the shrimp’s stomach triggered by Quorum Sensing (there must be a minimum number of bacteria in close proximity before the gene responsible for the toxin manufacture is ‘triggered’). Some reports of Biofloc ponds being very much less prone to EMS could be because Quorum Quenching bacteria in the microbial aggregates block Quorum Sensing and hence the release of the toxin.

After these cells die, the columnar hepatopancreal tubule cells begin to lose its attachment and ‘slough off’ into the GI tract. Extremely few shrimp diseases exhibit this characteristic ‘sloughing of hepatopancreal cells’ and is one of the major signs of EMS from a histology perspective. These cells can also disintegrate in a process called apoptosis leading to an accumulation of cellular debris that can provide substrate for secondary opportunistic bacteria (mostly Vibrio) and sometimes Gregarines. Such massive damage to the hepatopancreas leads to the death of the shrimp very quickly. The diagrams illustrate the mechanism of EMS/AHPNS as we understand it.

The bacteriophage attaches to the V parahaemolyticus and injects in phage DNA incorporating a virulence sequence from another bacteria.

The virulence sequence divides along with the bacteria but is triggered to produce the toxin when a Quorum is reached.

By the toxin (red) affecting the f-actin protein structure, cytoplasm flows out in ‘blebs’ seen in Transmission Electron Microscope (TEM).

Columnar hepatopancreas cells begin to ‘slough off’ into the lumen ending up in the GI tract.

Finally, secondary opportunistic bacteria and sometime gregarines feed off the cell debris of the dying hepatopancreas cells.

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