iSOLATION, Characterization And AUtogenous bACTERIAL vaccine development fOR a Chronic disease affecting brown-spotted grouper (Epinephelus chlorostigma)

Eng Khuan Seng *  and De Xun Tan
Biotechnology Applied Research Group
School of Chemical & Life Sciences
Nanyang Polytechnic
180 Ang Mo Kio Ave 8, Singapore 569830
Seng_Eng_Khuan@nyp.edu.sg

In recent years high demand for seafood protein has led to intensive aquaculture farming. As a consequence, this has led to an increase incidence of disease outbreaks especially the the farms located in tropical region like South-East Asia (SEA). Often, at times, farmers lose their fish stocks without knowing the causal of the fish deaths. Such was the case in a fish farm in Singapore. They were experiencing chronic mortality in their brown spotted grouper which could lead up to 50-60% mortality rate. The farmers did not know the cause of death of their juvenile groupers.

Here, we attempted to investigate the causal of the chronic deaths. Judging from an quick autopsy of infected fish we speculated that the grouper could have died from vibrio infection. Hence, using two agar plates, Thiosulfate - citrate - bile sucrose (TCBS) and Anacker & Ordal (AOA) agar, 34 and 24 bacterial isolates were discovered respectively. After dereplication, 31 isolates were chosen for further purification. From here, 48 samples were isolated and tested using a Universal Vibro PCR primers (VibAll), 23 were found to be positive for Vibrio. Of the 23, 17 was later found to be Vibrio harveyi using specific primer set. From the 17 positive V.harveyi samples, 4 different isolates were selected for gram staining, biochemical & physical characterization and results is as shown in Table 1. Following that prototype vaccines for all 4 isolates characterized was done by inactivating the bacterial using formalin. It was found that formalin at a concentration of just 0.5% (v,v) is sufficient to kill the bacteria. Using this formalin killed bacteria, a prototype vaccine was formulated. All 4 prototype V.harveyi vaccines are currently in the midst of being tested in the diseased affected fish farm. If the prototype vaccine is found to be successful, this would be the 1st autogenous Vibrio harveyi fish vaccine fully developed in Singapore to protect fish from Vibrio harveyi infections in local farms.