Study on the Efficacy of Pondguard in Improving Clinical Performance of White Leg Shrimp (Penaeus Vannamei) in an AHPND Bacterial Challenge Model

A bioassay trial was conducted to determine the efficacy of developed product, Pondguard as an anti-Acute Hepatopancreatic Necrosis Disease (AHPND) candidate. The Pondguard (Registration no. D 16060285-HBC) is consisting of natural oils, like, lavender oil, Eucalyptus oil and Pine oil. It maintains the immunity level of shrimp, which helps to protect shrimp from infectious diseases. Two doses of Pondguard i.e. 40 ppm and 80 ppm were selected for the trial. The treatment groups tanks were applied Pondguard whereas no application in control tanks throughout the experiment. The shrimp of both treatment and control were challenged by immersion method. The cumulative mortality reached up to 56.7% in positive control whereas 23.3 % in 80 ppm pondguard group and 13.3% in 40 ppm pondguard group at dpi 8 whereas no mortality recorded in negative control. The Relative Percent Survival of 80 ppm group was 64.7% and of 40 ppm group was 76.5%. The trial results show that the developed Pondguard has significant effect against AHPND-Vibrio parahaemolyticus in a controlled condition.

average weight was 0.5 g and average length was 4.33 cm. Prior to challenge average weight was 1.4 g.

Experimental system
Glass aquaria (30 L in volume) were used to set up the trial.
Each aquarium contained 25 L of seawater (15‰) with continuous aeration system and temperature was maintained around 26-30°C.
Experimental shrimp were stocked with the density of 40 individuals/aquaria (before challenge) and 15-20 individuals/aquaria (on challenge). The treatment and control groups were reared in 2 different rooms. The distance between two rooms was about 30 meters.

Feeding
The CP Prima, Indonesia for the trial, provided feed. The proximate composition of both feeds was the same. The feeding rate was 7% of MBW and the feeding frequency was 4 times/day (at 7AM, 11AM, 1PM and 7PM) throughout the trial period.

Pondguard
The CP Prima, Indonesia for the trial, provided Pondguard. The Pondguard is consist of Natural oils, like, lavender oil, Eucalyptus oil and Pine oil. It maintains the immunity level of shrimp, which helps to protect shrimp from infectious diseases. The Pondguard is registered under Ministry of Aquaculture, Indonesia with Registration no. D 16060285-HBC. The different concentration of Pondguard (40ppm, 80ppm) was applied to obtain the best dose to act against AHPND. The dose of Pondguard for the current trial was determined on the basis of previous study trial outcome. The de-termined dose of pondguard was started applying two days prior to challenge. It was applied on daily basis throughout the trial period.

Bacterial challenge
The bacterial strain used in this study was designated V36. This strain was originally isolated from shrimp diagnosed with AHPND histopathology (Strain was isolated from diseased shrimp collected in Soc Trang province in September 2016). Strain was stored at -80°C in TSB supplementing with 1.5% NaCl and 25% glycerol. The bacteria were identified as V. parahaemolyticus by green colonies on TCBS. Conventional API 20E biochemical tests and PCR with LTH primers [7]. Additionally, the isolate was positive on PCR with AP3 primers [8].
Bacterial culture were grown 24 h in TSB supplementing with 1.5 % NaCl at 28°C. Based on the standard curve determined for the strain. The OD corresponding to 10 8 cells ml-1 was diluted in seawater. Immersion method of challenge was applied in the experiment [9]. Shrimp were immersed for 15 minutes in this bacterial culture with continuous aeration and then both bacterial solution and shrimp were transferred to aquaria containing seawater reducing the bacterial concentration to 10 6 cells ml -1 . No water was exchanged for 48 hours after the challenge. Then 30% of water was renewed every 2 day.

Experimental design
Set up in 4 groups with 25L water. Challenge with V36 strain and added test products as detail in Table 1. Clinical signs observation and recording were followed up for 7 days post challenge.

Cumulative mortality
Mortalities were started at 9hrs of post challenge and seen clearly at 21-24hrs of post challenge. The average cumulative mortality are presented in Figure 2. Summary of cumulative mortality at 7 days after challenge from different treatment groups are compared and presented in ( Table   2). The lowest cumulative mortality was 13.3% in Treatment Group 1 (regular Feed + 40 ppm Pondguard+EMS).

RPS (Relative Percent Survival) on the basis of Cumulative Mortality (CM)
RPS (Relative Percent Survival) on the basis of Cumulative Mortality (CM)

TCBS plate counting
Comparison of total Vibrio count from water samples collected from different treatments of experiment is presented in Figure 3.

Discussion
The obtained laboratory results states that in a controlled environment Pondguard containing natural oil extracts provide pre-vention against AHPND. The treatment shrimp that challenged with AHPND/EMS V. parahemolyticus showed lower mortality rate as compared to positive control group. Furthermore, the obtained results and analysis gave a clear indication that we have identified a solution to combating EMS outbreaks in a controlled environment.
The AHPND gross-sign appearance in the challenged shrimp was similar and as described by [6,[10][11][12]. The rate of mortality in the challenged group was similar to the field reports i.e. heavy mortality for 3 to 5 days with significant drop in feed intake. The bioassay challenge trial conducted by [6] received the similar rate of mortality as recorded in the positive control of the current trial. The herbal origin products have proven to be anti-bacterial and anti-viral properties [13][14][15][16]. The next step would be conducting a field trial in the culture ponds [17][18][19][20][21].