22 INFOFISH International 4/2014 www.infofish.org Aquaculture E MS/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 Lin Ung Eng Huan