Acute Hepatopancreatic Necrosis Disease (AHPND), commonly known as Early Mortality Syndrome (EMS) is known to be caused by specific strains of Vibrio parahaemolyticus. This specific disease has severely affected the shrimp aquaculture industry across world. Among the several health additives developed, probiotics have been increasingly used in aquaculture to improve shrimp health as well as improve water quality by removing toxic metabolites. The objective of this trial was to evaluate the effectiveness of two Bacillus based probiotics on the disease tolerance of whiteleg shrimp when challenged with EMS- causing V. parahaemolyticus. Bacillus amyloliquefaciens CECT 5940 based product (Ecobiol®, 2E9 CFU/g) via feed application and B. subtilis based multi-strain product (AQUAVI® Pro-Pond, 1.5E10 CFU/g.) via water application were used for the experiment.
The trial comprised 7 groups including i) Negative control without EMS challenge (NC), ii) positive control challenged with EMS-causing V. parahaemolyticus (PC), iii) PC group treated with antibiotic (Cefotaxime via feed at 0.2% after the challenge (iv) PC group treated with probiotics (Ecobiol) via feed at 0.1% (v) PC group treated with probiotics (Ecobiol) via feed at 0.2% (vi) PC group treated with probiotics (Pro-Pond) via water at 0.4 ppm and (vii) PC group treated with probiotics via feed (Ecobiol) at 0.1% and via water (Pro-Pond) at 0.4 ppm. Water probiotics was applied once in every three days before the challenge and daily after the challenge whereas feed probiotic was applied daily throughout the experiment. Each treatment had 6 replicate tanks and each tank was initially stocked with 31 shrimps (0.57 ± 0.01 g, mean ± SD) at the density of 310 shrimps/m3. Shrimps were fed with the experimental diets and the water probiotic was also applied to the relevant treatment tanks for 21 days. On day 22 of the trial, shrimps in all groups except the NC were challenged with V. parahaemolyticus strain (LA-37) at the dose of 1E5 cfu/mL (LD50-96h) according to the calibration test results. Shrimp were observed for survival for next 10 days, i.e., 10-days post challenge period. After 12 h of the challenge, the first mortality by the AHPND was recorded and the mortality rate in the PC reached 50% after 108 hpc. At the end of the trial, the combined treatment of feed and water probiotics (0.1% Ecobiol + 0.4 ppm Pro-Pond) resulted in the highest survivability (61.6 ±8.4%), after the NC group which showed a survival rate of 95.45 ± 0.13%, both the treatments differed significantly from the PC. 0.2% Ecobiol® on feed also showed a relatively high survival rate (51.31±16.42%) and was the second highest among the treatment groups but not significantly different from the PC. According to the immunological assays, both of the test products, Ecobiol and Pro-pond, had improved immune response of shrimp. Among the treatments, the combined treatment (0.1% Ecobiol + 0.4 ppm Pro-Pond) showed the best immune response. Based on the results, combination of feed-based B. amyloliquefaciens CECT 5940 and water-based multi-strain B. subtilis products would be an effective strategy to improve the heath-status of whiteleg shrimp and protect against EMS-causing V. parahaemolyticus.