In Vivo Anti-Vibrio Evaluation of Sponge-Associated Bacteria on the Survival Rate of Litopenaeus vannamei Infected with Pathogenic Vibrio Species.

Sponge-associated bacteria are considered a rich source of bioactive compounds particularly to reduce the risk of Vibrio harveyi and Vibrio parahaemolyticus infection. The present study aimed to analyse the effectiveness of 19 isolates to control Vibrio infection in vivo. All 19 isolates displayed a non-pathogenic characteristic on shrimps (cell density of 106 cells/mL) as analysed using the pathogenicity test. The mortality caused by both Vibrio spp. on 50% of the shrimp population (LC50 value) had a cell density of 105 cells/mL as determined using the proportion interval method. On the basis of the challenge test, all isolates improved the survival rate of infected shrimps in diverse effectivities up to 89%, which was nearly 30% higher than the infected control. Two isolates coded as D6.9, and P5.20 reduced shrimp mortality after infection with Vibrio spp. 16S rRNA-based identification showed these isolates were closely similar to different genera of Bacillus and Staphylococcus. The extract derived from the most prospective isolate, D6.9, was dominated by 1-hydroxy-6-(3-isopropenyl-cycloprop-1-enyl)-6-methyl-heptan-2-one, hexadecanoic acid, 4-epicyclomusalenone [(24S)-24-methyl-28-norcycloart-25-en-3-one], and 2,4-dimethyl acetoacetanilide. This observation suggested these isolates characterised by in vivo anti-Vibrio activity need to be further developed as biocontrol candidates.

Immune response, gene expression, and intestinal microbial composition of Pacific white shrimp fed with multispecies synbiotic for the prevention of coinfection disease.

This study aimed to evaluate the application of synbiotic containing multispecies of probiotics with different cell densities in white shrimp rearing against infectious myonecrosis virus (IMNV) and Vibrio parahaemolyticus coinfection. This study used a completely randomized design with five treatments and three replications. One additional replication of each treatment was provided for the lethal sampling. Pacific white shrimp were fed with three dosages of synbiotic multispecies for 30 days, namely 103 CFU mL-1 (Sin 3), 106 CFU mL-1 (Sin 6), and 109 CFU mL-1 (Sin 9), and the controls without synbiotic administration consisted of the positive control (K +) and the negative control (K -). Pacific white shrimp from all treatments, except for the K - , were challenged with IMNV a dose of 100 µL and 106 CFU mL-1 V. parahaemolyticus, injected intramuscularly. Infected Pacific white shrimp showed clinical signs like anorexia, melanosis, empty gut, colorless hepatopancreas, and white necrotic areas in striated skeletal muscles, especially of the distal abdominal segments and uropod. The results showed that administration of synbiotic for 30 days resulted in higher immune parameters, such as total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RB), and total viable bacterial count (TBC) compared to K + /K - . After coinfection, they showed significantly higher levels for THC, PO, RB, gene expression prophenoloxidase (ProPO), and lipopolysaccharide and ß-1.3-glucan-binding protein (LGBP), better clinical signs, and lower mortalities. Sin 9 treatment significantly showed the highest survival rate (SR) compared to the other treatments.

Dietary supplementation of Pseudoalteromonas piscicida 1UB and fructooligosaccharide enhance growth performance and protect the whiteleg shrimp (Litopenaeus vannamei) against WSSV and Vibrio harveyi coinfection.

P. piscicida 1Ub and FOS were evaluated for their potential synbiotic effects on growth, immunological responses, and disease resistance against white spot syndrome virus and V. harveyi coinfection, the major pathogen in whiteleg shrimp aquaculture. Four different supplemented diets were used to feed the experimental shrimp for 40 days: control (control, no probiotic, and prebiotic), probiotic (PRO, P. piscisida 1UB 108 CFU mL-1), prebiotic (PRE, FOS 0.5% w/w), and the synbiotic (SYN, PRO + PRE). Shrimp's body weight, weight gain, specific growth rate, feed conversion ratio, survival, digestive enzyme activity, and metabolism-related gene expression were all evaluated on day 40. After 40 days, shrimp were infected with WSSV as the primary infection and V. harveyi as the secondary infection 24 h later. Shrimp were then grown for seven days and fed with a control diet. Survival, total hemocyte count (THC), differential hemocyte, phenol-oxidase (PO), respiratory burst activity (RB), and immune-gene expression were all analyzed at 0, 3, and 7 days after infection. The results showed that the PRO, PRE, and SYN supplementation improves whiteleg shrimp growth performance, immune responses, and protection against WSSV and V. harveyi coinfection. The increased activity of digestive enzymes and metabolism-related genes correlates with higher growth performance. The increase in THC, PO, RB, and immune-related gene expression after coinfection was associated with a significant reduction in shrimp mortality. Our findings also suggest that supplementing with synbiotics improves the overall performance of whiteleg shrimp significantly more than probiotics or prebiotics only.

Growth Performance and Intestinal Microbiota Diversity in Pacific White Shrimp Litopenaeus vannamei Fed with a Probiotic Bacterium, Honey Prebiotic, and Synbiotic.

This study aimed to evaluate the growth performance and intestinal microbiota composition in Pacific white shrimp after probiotic, honey prebiotic, or synbiotic treatment. Pacific white shrimp were treated for 45 days with probiotic (1% (v/w) of Bacillus sp. NP5 RfR probiotic), prebiotic (0.5% (v/w) of honey prebiotic), synbiotic (1% (v/w) of probiotic and 0.5% (v/w) prebiotic), or control (without addition of probiotic and prebiotic). Next-generation sequencing (NGS) was used to assess the effects of these treatments on growth performance and intestinal microbial diversity. The administration of a probiotic, prebiotic, or synbiotic led to increases in specific growth rate, feed conversion ratio, and digestive enzyme activities of amylase, protease, and lipase in Pacific white shrimp. The prebiotic treatment demonstrated the greatest effect, with values of growth rate of 3.09 ± 0.02 (% day-1), feed conversion ratio of 1.45 ± 0.00, and enzyme activities of 1.388 ± 0.0211 IU mg-1 protein for amylase, 0.055 ± 0.0004 IU mg-1 protein for protease, and 0.152 ± 0.0025 IU mg-1 protein for lipase. Analysis of the intestinal microbiota diversity revealed that prebiotic administration caused dominance of the phylum Bacteroidetes, whereas the probiotic and synbiotic treatments caused dominance of the phylum Proteobacteria. Moreover, prebiotic treatment was able to increase the diversity of Microbacterium, Lactobacillus, and Neptunomonas, which are established probiotic candidates in aquaculture. The probiotic, prebiotic, and synbiotic treatments induced a number of operational taxonomic units (OTUs) significantly higher than control treatment, that is, 470, 480, 451, and 344 OTU, respectively.

Effect in white shrimp Litopenaeus vannamei of Eleutherine bulbosa (Mill.) Urb. Powder on immune genes expression and resistance against Vibrio parahaemolyticus infection.

This study investigated the influence of Eleutherine bulbosa (Mill.) Urb. on the immune responses, bacterial population in the intestines, and resistance of white shrimp, Litopenaeus vannamei, against infection with Vibrio parahaemolyticus. Shrimp were fed with three dosages of powder, at 6.25 g kg-1 (P6.25), 12.5 g kg-1 (P12.5), and 25 g kg-1 (P25). One dosage of the crude extract was provided, 1.25 g kg-1 (E1.25), and the controls without administration of E. bulbosa consisted of a positive control (PC) and a negative control (NC). Feed supplementation was carried out for 30 days; then shrimp from all treatments were challenged by intramuscular injection with V. parahaemolyticus (106 cfu/mL), except for the NC. The results showed that supplementation with the powder and extract of E. bulbosa for 30 days resulted in significantly higher (P < 0.05) immune responses (total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RBs)), gene expression (prophenoloxidase (proPO), lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP)), and total bacterial count (TBC) compared to PC/NC. In post challenge testing, there were significantly higher levels for THC, PO, RBs, proPO, LGBP, and PE (peroxinetin), and the treatments were able to suppress V. parahaemolyticus in the intestines, hepatopancreas, and muscles and to reduce damage to the muscles and hepatopancreas. The survival rate with P12.5 was significantly higher compared to the other treatments. It was concluded that the shrimp receiving supplementation with the powder and extract of E. bulbosa had increased immunity and resistance against V. parahaemolyticus infection, with the best dosage being the P12.5 treatment.

Buton Forest Onion Extract (Eleutherine bulbosa (Mill.) Potential on Growth Performance of Vannamei Shrimp (Litopenaeus vannamei).

BACKGROUND AND OBJECTIVE: Vannamei shrimp Litopenaeus vannamei is an economically valuable aquaculture commodity but still faces problems due to disease and growth performance. Buton forest onion extract Eleutherine bulbosa (Mill.) Urbhas been shown can protect the shrimp from disease. This study aimed to evaluate the potential of the Buton forest onion extract on the growth of vannamei shrimp in tarpaulin pond. MATERIALS AND METHODS: The experiments were conducted for 90 days by adding Buton forest onion extract in feed at a dosage of 40 g kg-1. The Buton forest onion bulbs was extracted by maceration method using 96% ethanol. The test feed was made through the re-pelleting method. Treatment include supplementation of Buton forest onion extract diet supplemented with Buton forest onion extract diet at twice a week (treatment A) and control was without Buton forest onion extract(treatment K). Parameter measurement include final weight, average daily growth (ADG), feed conversion ratio (FCR), survival rate and water quality (temperature, salinity, turbidity and pH). RESULTS: This study was demonstrated the addition of Buton forest onion extract on feed with a frequency twice a week in significantly improve the growth performance of vannamei shrimp by improving the final weight and average daily growth at 65 and 90 days of maintenance. Water quality during the study was still within the optimum range for L. vannamei shrimp growth. CONCLUSION: This study showed that supplementation of Buton forest onion extract was able to improve the growth performance of shrimp in tarpaulin ponds.

Phytochemical analysis and antibacterial activities of Eleutherine bulbosa (Mill.) Urb. extract against Vibrio parahaemolyticus

To analyze compounds in Eleutherine bulbosa (E. bulbosa) (Mill.) Urb. extract and to determine its antibacterial capability against Vibrio parahaemolyticus (V. parahaemolyticus). Methods: E. bulbosa bulb extract was preprared using 96％ ethanol by the maceration method. Phytochemical investigation of E. bulbosa extract was analyzed using GC-MS, spectrophotometry and titrimetry methods. The zone of inhibition was identified by the diffusion agar method. The minimum inhibitory concentration and minimum bactericidal concentration were determined using the plate count method. The inhibitory rate against V. parahaemolyticus was determined by the microdilution method. Cellular leakage was evaluated by spectrophotometry and cellular damage was observed by scanning electron microscopy. Results: GC-MS analysis showed the high compound of the E. bulbosa extract was securixanthone E (7-hydroxy-1,2-dimethoxyxanthone). The compound groups also included fatty acid esters, isoquinolines, naphthalenes, and phenolics. The total phenolic content was (2.50 ± 0.00) mg/g, flavonoid (6.61 ± 0.00) mg/g, and tannin (0.03 ± 0.00)％. The greatest zone of inhibition and inhibitory rate were (11.83 ± 0.06) mm and (91.32 ± 2.76)％, respectively, at 10 mg/mL. The minimum inhibitory concentration was 0.156 mg/mL, while the minimum bactericidal concentration was 10 mg/mL. The E. bulbosa extract caused leakage and cellular damage to V. parahaemolyticus. Conclusions: The E. bulbosa extract possesses inhibitory activities against V. parahaemolyticus and causes cellular leakage and damage.