Potential synbiotic effects of a Bacillus mixture and chitosan on growth, immune responses and VP(AHPND) resistance in Pacific white shrimp (Litopenaeus vannamei, Boone, 1931).

The potential synbiotic effects of a Bacillus mixture and chitosan on growth, immune responses and disease resistance against Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) in Pacific white shrimp, were intensively investigated. Three effective strains of Bacillus amyloliquefaciens (A), Bacillus pumilus (P) and Bacillus subtilis (S) were mixed in pairs at a ratio of 5 × 108:5 × 108 CFU/kg diet and coated with the prebiotic chitosan (C) at a concentration of 20 mL/kg diet. Five different feed treatments were used to feed experimental shrimp for 5 weeks: control (control, no synbiotics), chitosan (coat, C) and the synbiotic treatments PAC, PSC and ASC. At week 5, the final length, final weight gain, weight gain, length, average daily gain, specific growth rate and feed conversion ratio, measured as growth parameters, were significantly upregulated in the PSC and ASC groups compared with the control and coat groups (P < 0.05). This result was consistent with the expression analysis of two growth-related genes (Rap-2a and GF-II) in the hepatopancreas and intestines of treated shrimp, as determined using qRT-PCR. The prebiotic chitosan and synbiotics PAC, PSC and ASC strongly induced significant differences in the expression of the Rap-2a and GF-II genes in the target organs compared with the expression in the control group at various time points (P < 0.05). Additionally, application of the synbiotic treatments also significantly enhanced the hepatopancreas characteristics and epithelial and intestinal wall thicknesses of the shrimp compared with the control. Interestingly, all the synbiotic treatments elevated phagocytic activity significantly at weeks 3 and 5 compared with that in the other groups. qRT-PCR analysis of immune-related genes also indicated that the prebiotic group and all synbiotic groups showed strong expression of anti-lipopolysaccharide (ALF) and prophenoloxidase (proPO) genes in the intestine. Finally, the synbiotic groups PAC, PSC and ASC exhibited stronger VPAHPND resistance at 120 h after exposure than the chitosan coat and control groups, with survival rates of 41.7 ± 11.55, 41.7 ± 0.00, 52.8 ± 5.77, 30.6 ± 15.28 and 22.2 ± 5.77%, respectively (P < 0.05). Based on the obtained information, all synbiotics were recommended for improved growth and immune responses, while ASC was the best for disease resistance against VPAHPND in Pacific white shrimp.

Probiotic effects of Bacillus spp. from Pacific white shrimp (Litopenaeus vannamei) on water quality and shrimp growth, immune responses, and resistance to Vibrio parahaemolyticus (AHPND strains).

Acute hepatopancreatic necrosis disease (AHPND), a serious disease caused by some Vibrio spp., impacts the Pacific white shrimp industry worldwide, especially in Thailand. To effectively overcome this problem, efficacious probiotic candidates were isolated from shrimp farms near coastal areas. The isolated Bacillus probiotics were screened for their ability to control pathogenic Vibrio spp. and various V. parahaemolyticus AHPND (VPAHPND) strains. Among the obtained probiotics, Bacillus subtilis AQAHBS001, which broadly inhibited various strains of VPAHPND, was evaluated on a laboratory scale in water-soluble and feed applications of viable probiotic. The water addition of 1 × 103-1 × 105 CFU/mL of this probiotic effectively reduced total ammonia but did not improve shrimp growth and resistance to VPAHPND. However, feed supplemented with the selected probiotic at 1 × 107 and 1 × 109 CFU/kg diet and provided to shrimp continuously for 5 weeks efficiently improved growth, as indicated by significant final weight gain, average daily growth, specific growth rates and feed conversion ratios. Additionally, this probiotic significantly elevated immune responses through phagocytic activity and clearance efficiency and enhanced the expression of the prophenoloxidase, lysozyme, and anti-lipopolysaccharide factor genes. Furthermore, B. subtilis AQAHBS001 obviously improved midgut characteristics by increasing microvilli and intestinal wall thickness. Finally, this probiotic evidently improved resistance to VPAHPND.