by Farshad Shishehchian, KritKhemayan, Zahra Javidi, Blue Aqua International Group of Companies, Thailand

 

Shrimp aquaculture has been dramatically affected by many pathogenic diseases, mainly caused by V. parahaemolyticus and White Spot Syndrome Virus (WSSV). The aim of this study was to evaluate the potential use of two functional feed additives of AlphaGuard*L Plus (Liquid) and AlphaGuard*P (Powder)composed of essential oils (eucalyptus, thyme, oregano belonging to the Myrtaceae and Lamiaceae family respectively), Medium Chain Triglycerides (MCTs) and natural bioactive compounds in shrimp against disease-caused pathogens, especially V. parahaemolyticus and WSSV.

The results indicated that AlphaGuard*L Plus effectively delayed disease progress in shrimp. These results suggest that the functional feed additive of AlphaGuard*P and AlphaGuard*LPLUS could be used in order to promote shrimp"s defense against pathogens.


Introduction

The practice of aquaculture intensification is impeded by health and nutrition affecting growth performance. To untangle these consequences, functional feed additives have been used to stimulate shrimp immune and improve shrimp performance specially to control viral and bacterial pathogensin recent treat shrimp diseases such as V. parahaemolyticus thatcaused Acute Hepatopancreatic Necrosis Disease (AHPND) and White Spot Syndrome Virus (WSSV) that caused White Spot Disease(WSD), combined with the implementation of biosecurity measures.

The composition of AlphaGuard composing MCT and essential oil are being recognised as GRAS practice. In this study, the efficiency of the AlphaGuard product was obtained by a combination of the data from disc-diffusion test results, disease challenges and histology, together with an evaluation of AlphaGuard product effects on the shrimp diseases.


Materials and methods

Pathogenic bacteria and viruses
Two virulent pathogens in shrimp, V. parahaemolyticus and V. harveyi in glycerol stock, have been sub-cultured in Tryptic Soy Broth (TSB) + one percentNaCl, incubated at 37oC for 24 hours, before streaked onto thiosulfate-citrate-bile salts-sucrose agar to obtain its pure culture.

WSSV suspension was prepared from the muscle of WSSV infected shrimp. Briefly, the WSSV-containing in shrimp muscle was removed from storage at -80°C and cut into uniform pieces under cold sterile conditions then homogenised in TN buffer (20 mM Tris-HCl, 400 mMNaCl, pH 7.4) at 0.1 g/ ml.

After centrifugation at 2,000 rpm for 10 minutes at 4°C, the supernatant was diluted to 1:100 with 0.9 percentNaCl and filtered through 0.45 micron. The resulting supernatant was stored at -80°C, until it was used as a source of WSSV injection for the challenge experiments.


The antimicrobial activity

Disc-diffusion test
5 µl of the of AlphaGuard*L Plus and AlphaGuard*P, concentration ranging from 0, 0.25, 0.5, 1.0 and 10 percent dilution with NaCl 0.85 percent, were absorbed into five millimetre diameter, 0.9 mm thick paper discs then air dried before being placed into 105 cells of bacteria culture in a petri dish 100 x 15 mm wide. 0.85 percentNaCl solution was used as negative control. Three replicate plates for each treatment were used and observations were recorded after 24 hours.


Artificial infection with V. parahaemolyticus and determining the number of Vibrio in shrimp hemolymph (HL) and hepatopancreas (HP)

V. parahaemolyticus was reactivated from storage at -80°C and cultured in TSB + one percentNaCl at 37°C overnight. The culture was centrifuged at 5,000 rpm for 10 minutes, to remove the supernatant, and the pellet was re-suspended in sterile 0.85 percentNaCl to a density of 8.4×106 CFU/ml.

The shrimp, on thefifth week, were infected by injecting 100 μL of the bacterial suspension into the second abdominal segment of healthy shrimp at fifth week of culture and mortality was recorded for seven days.

Shrimps were collected randomly on thethird day after infection for each treatment and washed three times with sterile water. HL was withdrawn from the pericardial cavity using a one-mL sterile disposable syringe under sterile conditions and added to an equal volume of sterile anticoagulant (adding 10 mM EDTA-Na2 to 450 mMNaCl, 10 mMKCl, 10 mM HEPES, pH 7.3, 850 mOsm.kg-1 or10 mM Tris-HCl, 250 mM Sucrose, 100 mM Sodium citrate, pH 7.6).

The HL and ground HP were serially diluted 10-fold with cold sterile PBS. Each dilution was spread on TCBS agar plates placed upside down in a 37°C incubator and cultured for 16–20 hours. Plates containing 30–300 bacterial colonies were counted, then the numbers were recorded as CFU/ml or CFU/g.


Artificial infection with WSSV

ml of the filtrate was injected intramuscularly into healthy shrimp at the fifth week of culture. Shrimps were collected randomly six hours after infection for histopathology examinationand mortality f was recorded for seven days.


Shrimp growth conditions and experimental groups

Shrimp were purchased from local Thai farms. They were tested negative forV. parahaemolyticus-caused AHNPD and WSSV by PCR analysis. After having acclimatised for one week in an aquarium, prior to the experiment, apparently healthy shrimp with uniform body length were divided randomly into three groups with six replicates per group, 20 shrimps per replicate.

The aquarium capacity was 100 litres, containing 60 litres of sea water. Saline water property was maintained at 15 ppt, pH 7.7-8.0 and DO>4.0 mg/L. Each treatment contains juvenile shrimps with an average initial weight of 2.6 g randomly stocked in each tank.

Shrimps were fed to satiation up to around 2.5-3 percent of their body weight, three times per day for five weeks. Feeding was adjusted daily, according to their ingested rate, to make sure that feed was totally consumed. Before feeding, molts, feces, and dead shrimps were removed, 20 percent of the water in each tank was exchanged every three days by new seawater.


Experimental diets and data collection

All groups were kept, during the experiment, under the same conditions as acclimation. The control group was fed by commercial shrimp pellets, coated with one percent chitin-chitosan. Treatment groups were fed by AlphaGuard*L Plus sprayed on or AlphaGuard*P, mixed with commercial shrimp pellets and coated by one percent chitin-chitosan, at dosage 5.0 ml or g/Kg feed. Mortality and water quality parameters were recorded daily. At the end of the trial, the survival rate was evaluated.


Statistical analysis

Statistical analysis experimental units, tanks, and aquariums were distributed in a completely randomised way. Quantitative data was checked for normality and homoscedasticity. Data wasanalysed using one-way analysis of variance (ANOVA) to search for significant (p < 0.05) differenceamong treatment means.


Results

The antibacterial activity by disc-diffusion methods and inclusion in shrimp diet on disease resistance of AlphaGuard*L Plus or AlphaGuard*P


Disc-diffusion test

The concentrations of AlphaGuard*L Plus and AlphaGuard*P at least one percent inhibit both V. harveyi and V. parahaemolyticus, however, there was higher inhibition efficacy for V. parahaemolyticus compare than V. harveyi. The effective concentration of AlphaGuard might have be lowered if we diluted in lipid solubles, such as ethanol, for testing.

Essential oil compositions, assay techniques and the mode of action have been intensively reviewed.This study did not investigate at molecular detail for its mechanisms, however, the main effect of bacteriocidal properties might be related to their interference of membrane integrity and permeability as the lipophilic compound from essential oil and MCTs.

Different fatty acids in MCTs have a different minimum inhibitory concentration (MIC), depending on the type of fatty acid, microorganism, and environmental pH. The synergistic and antagonistic aspect between ingredients in AlphaGuard is not elucidated in this study. The essential oil in AlphaGuard also acts as an antioxidant to prevent or slow down oxidation of unsaturated MCTs to prolong its shelf life besides anti-stress in shrimp.


V. parahaemolyticus challenge test

At the end of the feed trial experiment, shrimps were challenged by V. parahaemolyticus. Cumulative mortality rate was plotted out (see figure two). The results of mortality rate, after the V. parahaemolyticus challenge for a seven-day interval, showed that groups of shrimp fed by AlphaGuard*P and AlphaGuard*L Plus had significantly lower mortality rates than compared to the control group after day four of infection.

Both AlphaGuard*P and AlphaGuard*L Plus had the same statistical mortality rate after day five of infection. At the end of experiment, in contrast, the survival rate was not statistically correlated to mortality rate for AlphaGuard*P, (see figure three).

The ability of shrimp clearance from Vibrio spp. after three days of infection was plotted, (see figure four). The results indicated that the Vibriospp. count in the hemolymph of shrimp fed by AlphaGuard*P and AlphaGuard*LPlus were lower than the controlled groups. The ability of shrimp to defend against bacteria in HP, fed by AlphaGuard*LPlus, had also a much better ability to defend than shrimp not fed AlphaGuard.


WSSV challenge test

WSSV challenge test was conducted after five weeks of the feeding trial. The cumulative mortality was plotted, (see figure five). The group of shrimp fed by AlphaGuard*L Plusshowed significant delay in mortality, compared to the groups of control and AlphaGuard*P-fedshrimpduring their second, third and fourth days, (see figure six).

This is coincided with no histopathological sign of H&E staining WSSV infection tissue at the area of injection, except the generalisation of muscle necrosis that is likely related to the shrimp defense mechanism after three days of infection. Nevertheless, all groups ended up with 100 percent mortality.


Conclusion

The results of this trial suggested that the application of AlphaGuard*P and AlphaGuard*L Plus feed additive at 0.5 percent with commercial feed proved the efficacy on promoting the shrimp defense against pathogen. AlphaGuard*L Plusespecially displayed a better performance.

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