Novel indigenous probiotic isolated from the healthy Pacific white shrimp Litopenaeus vannamei intestine in differing stages based on metagenomic and screening approaches.

The healthy intestinal microbiota of shrimp can be used as an indicator sustainable shrimp production. In this study, the integrated of metagenomic and screening probiotic approach from healthy Litopenaeus vannamei intestines in differing stages was studied to find novel indigenous probiotics. The microbiota from intestine of naupli, post larva (PL-10), juvenile (40 days), and adult (80 days) of Pacific white shrimp were characterized using a high-quality sequence of V3-V4 of 16S rRNA gene as the hypervariable region. The classifiable sequence number was detected in 54 phyla. Several core intestine bacteria, 35 of these 557 genera, have a prevalence >10 sequences across all samples. We found microbiota were different taxa in the difference stages, such as Proteobacteria, Firmicutes, and Bacteriodetes. The top 10 most abundant genera were Vibrio, Pseudoalteromonas, Spingomonas, Marinibacterium, Klebsiella, Alteromonas, Aestuaribacter, Shimia, Stenotrophomonas, and Ruegeria. Microbiota profiling based on a metagenomic approach was integrated with screening assessment for pathogenicity, antagonistic activity with Vibrio parahaemolyticus Vp5, antibiotic resistance, and digestive enzyme activities. As their assessment activity, several screened culturable bacteria were 19 of these 84 isolates. Three isolates with high activities (P < 0.05) found as novel indigenous probiotics were Shewanella algae A1, Shewanella algae A3, and Vibrio diabolicus UB3. Integrating metagenomic and screening methods was a new signature for the isolating novel indigenous probiotics in Pacific white shrimp.

Protective effects of immersion immunization of koi with Escherichia coli DH5α carrying DNA vaccine against koi herpesvirus.

This study aims to immunize the koi fingerlings immersion using the formalin-killed and freeze-dried E. coli DH5α carrying plasmid for the KHV DNA vaccine. 200 fish on each tank in a total water volume of 20 L. Each tanks consists of different vaccination group: PBS as control (10 mL; C), empty E. coli DHα (10 mL at 108 CFU mL-1; E), formalinkilled E. coli DHα:ORF81 (10 mL at 108 CFU mL-1; KE), freezedried E.coli DHα:ORF81 without formalin inactivation (10 mL at 108 CFU mL-1; FE), and formalin-killed and then freeze-dried E. coli DHα:ORF81 (10 mL at 108 CFU mL-1; KFE). The bath vaccination was conducted for 1 × 30 min. For the challenged test, fish were immersed with the 100 mL of LD50 dose of KHV (10-2 dilution from the KHV stock) for 30 min. The vaccination using E. coli DH5α:ORF81 could significantly modulate the innate and adaptive immunity of the fish and result in higher fish survival after KHV infection. The vaccination using formalin-killed or formalin-killed and freezedried E. coli DH5α:ORF81 will be further developed as an alternative to the costefficient koi or carp fingerlings vaccination method.

High carbohydrate increases amylase, plasma glucose, and gene expression related to glycolysis in giant gourami Osphronemus goramy.

The present study elucidated hepatic molecular and physiological responses of giant gourami to high dietary carbohydrates. Two levels of dietary carbohydrate, normal carbohydrate (34%, NC) and high carbohydrate (53%, HC), were offered to the fish for 60 days. We evaluated the expression of genes that are related to carbohydrate metabolism, lipogenic capacities, amino acid catabolism, Krebs cycle, and energy sensing. In addition, we also observed the digestive enzyme activities, plasma glucose, glycogen content, whole-body composition, and growth performance of the fish. On day 30 after treatment, fish fed with high dietary carbohydrate level has significantly higher expression of gck, pk, hk, and ldh than the NC group (P < 0.05). In contrast, fish in the HC group had lower expression of irs1, igf-1, sdh, fbp, g6pc, gyp, and ampk compared to the NC fish (P < 0.05). On day 60 of the feeding trial, gck and hk expressions were still higher in the HC group (P < 0.05), and gyp, gdh, and ampk became increasingly expressed in the HC group. The increase of dietary carbohydrates resulted in significant increases in amylase and protease activity, plasma glucose, liver glycogen, crude protein, and lipid contents of the fish whole-body (P < 0.05). The high carbohydrate feeding reduced the fish growth rate but increased feed efficiency and did not affect mortality. In conclusion, giant gourami could utilize high carbohydrates due to a high amylase secretion, high modulation of carbohydrate metabolism, and large glucose storage capacity.

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.

Development of primary cell culture from spleen of giant gourami Osphronemus goramy for propagation of giant gourami iridovirus (GGIV).

The severe mortality of fish due to the infection of megalocytivirus caused significant economic losses. Since 2011, megalocytivirus (giant gourami iridovirus (GGIV)) has become the main pathogen in giant gourami (Osphronemus goramy), particularly in West Java, Central Java and Bali. This study aimed to develop primary cell culture from spleen as the target organ for propagating megalocytivirus in vitro, which was developed by explant method with enzymatic dissociation. Optimization was carried out at incubation temperature, medium and serum concentrations. The origin of the primary cell, cell susceptibility and GGIV pathogenicity were observed. The results showed that the primary cell (GP cells) can grow well in 10% foetal bovine serum L-15 medium at 27°C, which was sufficient for cell growth. PCR and BLAST analyses showed the primary cell was originated from giant gourami. In infected GP cells, cell enlargement and cell rounding were observed. Virus propagated in GP cells was highly virulent when injecting giant gourami in an artificial infection experiment. Intraperitoneal injection of diluted virus supernatant showed 100% mortality in 7-11 days post-injection and 97% mortality in 21 days post-cohabitation, with abnormalities observed in spleen and kidney. In conclusion, GP cell was successfully subcultured for more than 30 passages and susceptible to GGIV.