Expression of recombinant phytase of Bacillus subtilis E20 in Escherichia coli HMS 174 and improving the growth performance of white shrimp,Litopenaeus vannamei,juveniles by using phytase‐pretreated soybean meal‐containing diet

To produce a thermostable and neutral phytase (phy) of Bacillus subtilis E20 in Escherichia coli HMS174 and evaluate its efficiency in improving growth performance. The phy C of B. subtilis E20 was expressed in E. coli HMS 174, and then the 42‐kDa recombinant phy C was purified by Ni‐NAT and analysed by SDS–PAGE. The recombinant phy C had optimal ranges of pH of 6 ~ 7 and temperature of 50 ~ 60 °C. A thermostability analysis showed that the enzyme is a thermostable phytase, and around 33% of residual activity was detected after being incubated at 90 ~ 100 °C for 10 min. The recombinant phy C‐pretreated soybean meal for feed preparation improved white shrimp, Litopenaeus vannamei, growth and feed efficiency. Overall, the neutral and thermostable phy C is suitable for aquafeed, and it is able to improve the nutritional utilization, resulting in enhanced shrimp growth and reduced feed costs.     

Dietary supplementation with Bacillus subtilis LT3‐1 enhance the growth,immunity and disease resistance against Streptococcus agalactiae infection in genetically improved farmed tilapia,Oreochromis niloticus

A feeding trial was conducted to investigate the effect of different levels of Bacillus subtilis LT3‐1 in diets on growth, immune parameters, intestinal morphology and disease resistance in genetically improved farmed tilapia, Oreochromis niloticus. Fish (46.91 ± 0.17 g) were fed with a basal diet supplemented with B. subtilis LT3‐1 at 0 (B0), 3.8 × 10¹⁰ (B1), 7.6 × 10¹⁰ (B2), 1.14 × 10¹¹ (B3) and 1.52 × 10¹¹ (B4) CFU kg^?1 for 6 weeks. The results showed that the weight gain of fish in B1 group was significantly enhanced compared to that in B0 group (p < 0.05). The addition of B. subtilis significantly affected serum biochemical indices (total protein, albumin, aspartate aminotransferase, alkaline phosphatase). Besides, the haematocrit, total counts of red and white blood cells, as well as the serum catalase and lysozyme activities, were increased, whereas the serum malondialdehyde, the serum immunoglobulin M and complement three contents were reduced. Parameters for intestinal morphology suggested a healthier intestine for the fish fed B. subtilis‐supplemented diets than fish fed the control diet. The survival rate after Streptococcus agalactiae challenge increased in tilapia fed with B. subtilis. The present study demonstrated B. subtilis can effectively improve growth, immunological status and resistance against S. agalactiae infection in tilapia farming.     

Using a fermented mixture of soybean meal and earthworm meal to replace fish meal in the diet of white shrimp,Penaeus vannamei (Boone)

An experiment was conducted to evaluate the potential of a Bacillus subtilis E20‐fermented mixture (FSFEM) containing soybean meal (SBM) and Eisenia fetida earthworm meal (EM) at a ratio of 4:1 to increase the methionine level in order to satisfy the methionine requirement of white shrimp, Penaeus vannamei in a diet with fish meal (FM) completely replaced by mixtures. B. subtilis E20 fermentation improved the mixture's palatability and utilization based on better growth performance in comparison to shrimp fed FSEM (contains fermented SBM and EM at a ratio of 4:1) diets. FSFEM is a good substitute for FM. Maximal replacement levels of FM with FSFEM were 80% in a shrimp diet with 37% of crude protein and 7% of crude lipid based on weight gain and 100% based on feeding efficiency. In addition, shrimp fed experimental diets had no significant differences in survival after being challenged by Vibrio alginolyticus. It is suggested that B. subtilis E20‐FSFEM has the potential to replace FM in cultured shrimp diets.     

Effects of dietary supplementation of a marine thermotolerant bacterium,Bacillus paralicheniformis SO‐1, on growth performance and immune responses of Nile tilapia,Oreochromis niloticus

This study was carried out to evaluate the effects of dietary supplementation of thermotolerant bacterium on growth and immune responses of Nile tilapia (Oreochromis niloticus). Bacillus paralicheniformis SO‐1 was isolated from marine environments and incorporated into four isonitrogenous (300g/kg crude protein; cp) and isocaloric (18 MJ/kg) diets at four concentrations: 0, 5, 10 and 20 g/kg diet. Each diet was fed to triplicate groups of Nile tilapia (41.5 ± 0.5 g average weight) at a daily rate of 3% of their biomass, three times a day for 50 days. At the end of the feeding trial, the growth rates, feed utilization efficiency (feed conversion ratio, protein efficiency ratio, protein productive value), digestive enzymes (protease, amylase and lipase) activities, immunological response (serum lysozyme activity, phagocytic activity, respiratory burst and superoxide dismutase activity) and the expression of immune‐related genes [interleukin‐1 (IL‐1), interleukin‐4 (IL‐4) and interleukin‐12 (IL‐12)] were determined. Growth rates, digestive enzymes activities and immunological parameters were significantly improved (p < 0.05) with increasing supplemental SO‐1 up to 10 g/kg. However, further increase in bacterial concentration to 20 g/kg lead to significant decline in fish performance and immune response (p < 0.05). The expression of IL‐1, IL‐4 and IL‐12 genes was significantly up‐regulated (p < 0.05) in the liver of Nile tilapia fed SO‐1‐treated diets. This study clearly demonstrated that B. paralicheniformis SO‐1 could be considered as an efficient growth promoter and immune‐stimulating probiotic for farmed Nile tilapia.     

Beneficial effects of Bacillus subtilis on water quality,growth, immune responses,endotoxemia and protection against lipopolysaccharide‐induced damages in Oreochromis niloticus under biofloc technology system

The present research explored the effects of Bacillus subtilis on water quality, growth, immune responses, endotoxemia and protection against lipopolysaccharide (LPS) damages in Nile tilapia Oreochromis niloticus under biofloc system. B. subtilis was added at 0, 1, 2, 3 and 4 grams (1.19 × 10⁸ CFU/g) per kg of basal diet, named T1 (control), T2, T3, T4 and T5, respectively, and fed to fish (14.82 ± 0.42 g) for 50 days. The concentrations of TAN, NO₂ and NO₃ were significantly reduced, and fish fed probiotics displayed significantly better growth performances versus the control, concomitantly with significantly enhanced activities of digestive enzymes. They also showed significantly declined serum glucose and cholesterol vice versa significantly improved immune responses (total protein, albumin, globulin, lysozyme, alternative complement, protease, immunoglobulins, alkaline phosphatase and respiratory burst), antioxidant capacity (superoxide dismutase, total antioxidant capacity, malondialdehyde) and skin mucus parameters (total protein, lysozyme, alternative complement, protease, immunoglobulins). Meanwhile, significantly lower endotoxin (LPS) concentrations were detected in the intestines and serum of fish fed probiotics. LPS challenge induced profound oxidative stress and impaired immune responses. Interestingly, probiotic alleviated LPS‐induced damages and restored mentioned parameters. In conclusion, B. subtilis effectively enhanced fish production, immunity and protection against LPS‐induced damages in tilapia under biofloc system.     

Comparison of Edwardsiella ictaluri isolates from different hosts and geographic origins

The intraspecific variability of E. ictaluri isolates from different origins was investigated. Isolates were recovered from farm‐raised catfish (Ictalurus punctatus) in Mississippi, USA, tilapia (Oreochromis niloticus) cultured in the Western Hemisphere and zebrafish (Danio rerio) propagated in Florida, USA. These isolates were phenotypically homologous and antimicrobial profiles were largely similar. Genetically, isolates possessed differences that could be exploited by repetitive‐sequence‐mediated PCR and gyrB sequence, which identified three distinct E. ictaluri genotypes: one associated with catfish, one from tilapia and a third from zebrafish. Plasmid profiles were also group specific and correlated with rep‐PCR and gyrB sequences. The catfish isolates possessed profiles typical of those described for E. ictaluri isolates; however, plasmids from the zebrafish and tilapia isolates differed in both composition and arrangement. Furthermore, some zebrafish and tilapia isolates were PCR negative for several E. ictaluri virulence factors. Isolates were serologically heterogenous, as serum from a channel catfish exposed to a catfish isolate had reduced antibody activity to tilapia and zebrafish isolates. This work identifies three genetically distinct strains of E. ictaluri from different origins using rep‐PCR, 16S, gyrB and plasmid sequencing, in addition to antimicrobial and serological profiling.     

Effects of two dietary probiotics (Bacillus subtilis or licheniformis) with two prebiotics (mannan or fructo oligosaccharide) in Japanese eel,Anguilla japonica

A 12‐week feeding trial was conducted to investigate the effects of two dietary probiotics; Bacillus subtilis KCTC 2217 or Bacillus licheniformis KCCM 11775 with two prebiotics; mannan oligosaccharide (MOS) or fructooligosaccharide (FOS) in Japanese eel. Fish averaging 12.8 ± 0.47 g (mean ± SD) were randomly distributed into five treatments with triplicate tanks. A basal control diet (CON) and four synbiotic diets supplementing B. subtilis + MOS (BSM), B. subtilis + FOS (BSF), B. licheniformis + MOS (BLM), and B. licheniformis + FOS (BLF). Weight gain and specific growth rate of fish fed all synbiotic diets were higher than those of fish fed CON. Immune‐related gene expression of heat shock protein 70 and immunoglobulin M of fish fed BSF and BLM were significantly higher than those of fish fed CON. Fish fed BSF and BLM had significantly higher intestinal villi length than those of fish fed BLF and CON (p < .05). Disease resistance against Aeromonas hydrophila of fish fed all synbiotic diets were significantly higher than those of fish fed CON (p < .05). Therefore, these results indicated that dietary B. subtilis with FOS (BSF) and B. licheniformis with MOS (BLM) could have beneficial effects on intestinal morphology, and immune‐related gene expression in Japanese eel.     

Effects of diets supplemented with either individual or combined probiotics,Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, on the immune response and disease resistance of the mud crab,Scylla paramamosain (Estampador)

Two trials, including firstly, diets incorporating individual or combined species of probiotics, Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, were fed to the mud crab, Scylla paramamosian, for 28 days to evaluate the growth, immunity and disease resistance, and secondly, the optimal level of B. subtilis E20 in the diet by tracking the growth, immunity and disease resistance after 28 days of feeding were analysed. No significant differences in growth, total haemocyte counts, respiratory bursts, superoxide dismutase, or glutathione peroxidase were found in the two trials. Crabs fed the B. subtilis E20‐containing diet (D20) had significantly increased phenoloxidase (PO) activity, phagocytic activity (PA) and disease resistance compared with crabs fed the control and the mixed probiotics diet (MD). The mortality of crabs fed the D20 was significantly lower than that of crabs fed the L. plantarum 7‐40‐containing diet. L. plantarum 7‐40 had a great inhibitory effect on the growth of B. subtilis E20, which may have led to the decreased probiotic effect of the MD. An analysis of the optimal level of B. subtilis E20 in the diet showed that crabs fed the B. subtilis E20‐containing diet at a level of 10^9–10 cfu kg^?1 had better immune response of PO and PA, and disease resistance in the second trial.     

Significant association of SNP polymorphism in the tilapia enhancer of polycomb homolog 1 gene with salt tolerance

Identifying candidate genes involved into osmoregulation provides a basis for developing molecular markers for breeding of saline tilapia. In this study, we characterized and conducted a functional analysis of the Enhancer of Polycomb Homolog 1 (EPC1) gene in Nile tilapia. The length of the EPC1CDS sequence was 1161 bp, including 14 exons encoding 386 amino acid residues. The expression for EPC1 was investigated in the gill, brain and intestine tissues of Nile tilapia that challenged by 0 ppt, 10 ppt, 15 ppt and 20 ppt of salinity content by qRT‐PCR. We found that the gene was significantly down‐regulated at 20 ppt of high salinity stress. We also detected significant evidence of 5 SNP association in the EPC1 gene with salt tolerance trait by genotyping 192 extreme individuals from a full‐sib tilapia family (N = ~500). The individuals with heterozygous SNP genotypes in the population (with an average survival time of 3,064 s) were significantly less tolerant than the other individuals with the homozygote genotypes (with an average survival time of 5,986 s). Further functional analysis on the EPC1 protein sequences from 31 fish species inhabiting different salinity environments identified seven amino acid sites as significantly associated sites (α < 0.01) with salinity content. These data suggested that the EPC1 gene may be a candidate gene related to osmoregulation process in tilapia. Our findings could contribute to selection of the saline tilapia by using marker‐assisted selection technique.     

Effect of combination of dietary Bacillus subtilis and trans‐cinnamic acid on innate immune responses and resistance of rainbow trout,Oncorhynchus mykiss to Yersinia ruckeri

The present study investigated the effects of combination of dietary Bacillus subtilis and trans‐cinnamic acid on serum biochemical parameters, innate immune responses and resistance of rainbow trout, Oncorhynchus mykiss to Yersinia ruckeri. Six experimental groups of fish with mean weights of 20.58 ± 0.35 g were used in the study. Five experimental groups of fish were fed diets containing Bacillus subtilis (10⁷ per gram) or a mix of the Bacillus subtilis (BS) and trans‐cinnamic acid (25 mg/kg‐25trcBS, 50 mg/kg‐50trcBS, 75 mg/kg‐75 trcBS, 150 mg/kg‐150 trcBS), whereas an additive‐free basal diet served as the control (Cont). In this study, an increase was observed in granulocyte percentage, respiratory burst activity, phagocytic activity, phagocytic index, myeloperoxidase activity and total antiprotease activity especially in fish fed with mix of the BS and trans‐cinnamic acid‐supplemented diets (p < .05). Moreover, at the end of the 20‐day challenge period the survival rates and antibody titre (p < .05), and relative per cent survival were higher in the BS group and all trcBS groups compared with control group. As a conclusion, the results in the present study show that feeding rainbow trout with diets containing a mix of B. subtilis and trans‐cinnamic acid over a 60‐day period might be sufficient for improving fish immune responses and disease resistance against Y. ruckeri.     

Combined effects of dietary malic acid and Bacillus subtilis on growth,gut microbiota and blood parameters of Nile tilapia (Oreochromis niloticus)

The study investigated effect of dietary supplementation with malic acid, Bacillus subtilis and a mixture of the two (3 × 2 factorial trial) on Nile tilapia (Oreochromis niloticus) health. Treatment groups (T1–T6) were fed diets containing three levels of malic acid (0.0, 5.0 and 10.0 g/kg), each of which was supplemented with 0 and 1.1 × 10⁵ cfu/g B. subtilis, respectively. Each treatment group was assigned randomly to triplicate groups of 35 fish (5.26 ± 0.06 g) in 18 tanks for 84 days. The results indicated that survival was higher in all dietary treatments as compared to the control fed fish. The highest values of FBW, WG and SGR (%), PER, PPV and ER were recorded in groups T4 and T6, which were fed diets supplemented with 5 g malic acid/kg and 1.1 × 10⁵ cfu/g B. subtilis and 10 g malic acid/kg and 1.1 × 10⁵ cfu/g B. subtilis, respectively. The best value of FCR was obtained in groups T4 and T6. The lowest total bacterial count in the gut and faeces was detected in fish from group T6. The values of haematocrit, haemoglobin, red blood cells, white blood cells, total protein, albumin and globulin were significantly higher (p < .05) in fish from groups T4 and T6 (diets supplemented with malic acid and B. subtilis). As compared to the control fed fish, te mixture of these substances are promising as immune enhancher in aquacultured fish.     

Evaluation of growth and fatty acid profile of Nile tilapia (Oreochromis niloticus) fed with Schizochytrium sp.

The objective of this study was to evaluate growth, body chemical composition and lipid profile of Nile tilapia juvenile fed with Schizochytrium sp. Two hundred and forty Nile tilapia (Oreochromis niloticus) juvenile (1.33 ± 0.11 g) were distributed in 20 aquariums, at the density of 12 fish per aquarium. The juvenile were fed with five levels of Schizochytrium sp.: Control (0 g/kg of Schizochytrium sp. in feed); AS10 (10 g/kg of Schizochytrium sp. in feed); AS20 (20 g/kg of Schizochytrium sp. in feed); AS30 (30 g/kg of Schizochytrium sp. in feed) and AS40 (40 g/kg of Schizochytrium sp. in feed). The inclusion of Schizochytrium sp. increase the body weight, weight gain, final biomass and biomass gain of tilapia juvenile. The body crude protein of tilapia was increased after addition of microalgae. Juvenile lipid profile also was influenced when fed with 40 g of Schizochytrium sp. per kg and have it levels of omega‐3 and docosahexaenoic acid (DHA) increased, and omega 6:3 ratio decreased. Tilapia juvenile fed with Schizochytrium sp. per kg have better growth, omega‐3 and DHA levels rising in the body, being a considerable source of fatty acids for human nutrition. Levels above 20 g of Schizochytrium sp. per kg in the feed favour the increase of protein in Nile tilapia body.     

Intestinal microbiota of white shrimp,Litopenaeus vannamei,fed diets containing Bacillus subtilis E20‐fermented soybean meal (FSBM) or an antimicrobial peptide derived from B. subtilis E20‐FSBM

The purpose of the present study is to profile the microfloral structure in the intestines of the white shrimp, Litopenaeus vannamei, after being fed a control diet, in comparison with the diets containing Bacillus subtilis E20‐fermented soybean meal (FSBM) or an antimicrobial peptide (AMP) isolated from B. subtilis E20‐FSBM (diets designated FSBMD and AMPD, respectively) for 60 days. Metagenomic data showed detection of eight phyla, 20 classes, 40 orders, 68 families and 96 genera. Despite no statistically significant difference, an evaluation of microbial diversity recorded higher species richness, Shannon–Weaver diversity index and evenness in the AMPD and FSBMD groups, compared to the control diet group. Venn diagrams showed that 58 of the operational taxonomic units (OTUs) were shared among the control, FSBMD and AMPD groups, but additional beneficial microbes were only found in the FSBMD and AMPD groups. In addition to the augmentation of beneficial bacteria in the FSBMD and AMPD groups, the abundance of potential pathogens, Vibrio and Flavobacterium, was lower in the gut of shrimp fed the FSBMD and AMPD. The results clearly suggest that the FSBMD and AMPD have the capability to change the microfloral structure of shrimp intestines and could be used for disease prevention in shrimp aquaculture.     

Dietary supplementation of garlic (Allium sativum) modulates gut microbiota and health status of tilapia (Oreochromis niloticus) against Streptococcus iniae infection

This study was conducted to characterize the causative agent of streptococcosis in tilapia (Oreochromis niloticus) and control of Streptococcus infection by means of garlic (Allium sativum) supplementation. The morphological, biochemical and polymerase chain reaction amplification confirmed 11 isolates belong to Streptococcus iniae from the infected fish eyes and tissue samples. Random screening of 12 well‐known medicinal plant parts against S. iniae revealed the garlic extract as the most effective herbal recovery against Streptococcus infection. In vivo challenge test with dietary supplementation of garlic powder significantly improved survival rates of fish against S. iniae infections, and modulate the microbial community and cytokine gene expression profiling in the intestine of the experimental tilapia. Among the two garlic supplemented treatments, 1.0 g garlic supplemented diet significantly increased (p < 0.05) the survival rates of tilapia and the gut bacterial operational transitional units abundance for Proteobacteria and Tenericutes, the phyla associated with healthy intestinal flora. The bacterial diversity index also found high with garlic supplemented diets. Significant upregulations of IL‐10 and IL‐17F gene expression in the intestinal tissue were observed with 1.0 g garlic supplemented diet where IL‐8 and IL‐1β expression levels were relatively static. The dietary supplementation of garlic, therefore, could be effective in the prevention of S. iniae infection in fish.     

Animal by‐products with or without enzymatic hydrolysis completely replacement of fish meal in genetically improved farmed tilapia diets (Oreochromis niloticus)

An 8‐week feeding trial was performed to examine the potential of total replacement of fish meal with animal by‐products with or without enzymatic components in juvenile genetically improved farmed tilapia (GIFT) diets, Oreochromis niloticus, (mean initial weight, 19.71 ± 0.28 g), reared in RAS system. Five isonitrogenous and isoenergetic diets were formulated with fish meal for the diets with total replacement of the isonitrogenous amounts of enzymatic fish meal (E‐FM), chicken liver meal (CLM), enzymatic chicken liver meal (E‐CLM), dried porcine solubles (DPS) and enzymatic dried porcine solubles (E‐DPS) respectively. The results indicated that the weight gain rate (WGR) and specific growth rate (SGR) in the FM group were significantly higher than the E‐FM and E‐CLM groups (p < .05), but the specific growth rate (SGR) in the E‐CLM group lower than the E‐FM group. The feed conversion ratio (FCR) in FM and CLM was significantly lower than the E‐FM, E‐CLM and E‐DPS groups (p < .05), and no significant difference was observed among the FM, CLM and DPS groups (p > .05). The values of the protein retention efficiency (PRE) and protein efficiency ratio (PER) in FM, CLM and DPS groups were significantly higher than the E‐CLM group (p < .05). The values of feed intake ratio (FIR) in FM and CLM groups were significantly lower than the E‐FM, E‐CLM and E‐DPS groups (p < .05). There was no significant difference in the whole body contents of moisture, crude lipid and crude ash among the FM and E‐CLM groups (p > .05). The whole body contents of crude protein in the FM group were significantly higher than the E‐FM, CLM, E‐CLM DPS and E‐DPS groups (p < .05). The apparent digestibility coefficients (ADC) of dry matter, crude protein and crude lipid in the FM, E‐FM, E‐CLM and E‐DPS groups were not significantly different (p > .05). The serum glucose(GLU), total cholesterol (TCHO) and triglycerides (TG), or the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were not affected by the different treatments (p > .05). There was no significant difference in serum total protein (TP) or albumin (ALB) and between the FM group and other groups (p > .05). There was no significant difference in the serum urea nitrogen (BUN) content in the FM, CLM, E‐CLM and DPS groups (p > .05). Fish fed with the CLM and DPS diets had significantly lower hepatic GHR1, IGF1 and IGF2 gene mRNA expression levels than in fish fed the FM diet (p < .05). The present data showed that 6% of dietary fish meal can be totally replaced by the chicken liver meal and dried porcine solubles with no effect on the growth performance of GIFT strain tilapia.     

Dietary ratios of n‐3/n‐6 fatty acids do not affect growth of Nile tilapia at optimal temperatures (28°C) nor at temperatures that simulate the onset of winter (22°C)

Nile tilapia (Oreochromis niloticus) juveniles were fed diets containing 13 g/kg total polyunsaturated fatty acids (PUFAs) at different n‐3/n‐6 dietary ratios (0.2, 0.5, 0.8, 1.3 and 2.9) for 56 days, at 28°C. Subsequently, fish were submitted to a winter‐onset simulation (22°C) for 33 days. PUFA n‐3/n‐6 dietary ratios did not affect fish growth at either temperature. At 28°C, tilapia body fat composition increased with decreasing dietary PUFA n‐3/n‐6. Winter‐onset simulation significantly changed feed intake. The lowest dietary n‐3/n‐6 ratio resulted in the highest feed intake. At both temperatures, body concentrations of α‐linolenic acid, docosahexaenoic acid, eicosatrienoic acid and docosapentaenoic acid decreased as dietary n‐3/n‐6 decreased. Body concentrations of eicosapentaenoic acid (EPA, 20:5 n‐3) increased with decreasing concentrations of dietary EPA. The n‐6 fatty acids with the highest concentrations in tilapia bodies were linoleic acid and arachidonic acid (ARA, 20:4 n‐6). At 28°C, SREBP1 gene expression was upregulated in tilapia fed the lowest n‐3/n‐6 diet compared to tilapia fed the highest n‐3/n‐6 ratio diet. Our results demonstrate that a dietary PUFA of 13 g/kg, regardless of the n‐3/n‐6 ratio, can promote weight gains of 2.65 g/fish per day at 28°C and 2.35 g/fish per day at 22°C.     

Virulence regulation of cel‐EIIB protein mediated PTS system in Streptococcus agalactiae in Nile tilapia

Streptococcus agalactiae is a major pathogen of tilapia causing significant economic losses for the global aquatic industry yearly. To elucidate the role of cel‐EIIB protein‐mediated phosphotransferase systems (PTS) in the virulence regulation of S. agalactiae, cel‐EIIB gene deletion in a virulent strain THN0901 was achieved by homologous recombination. The cellobiose utilization of △cel‐EIIB strain was significantly decreased relative to S.a.THN0901 strain incubating in LB with 10 mg/ml cellobiose (p < 0.05). The biofilm formation ability of △cel‐EIIB strain was also significantly decreased when cultured in BHI medium (p < 0.05). Under a lower infection dose, the accumulative mortality of tilapia caused by △cel‐EIIB strain was dramatically decreased (20%), of which S.a.THN0901 strain and △cel‐EIIB::i strain were 53.33% and 50%, respectively. The competition experience using tilapia model indicated the invasion and colonization ability of △cel‐EIIB strain was significantly weaker than that of S.a.THN0901 strain (p < 0.05). Compared to △cel‐EIIB::i strain, the mRNA expression of csrS, csrR, rgfA, rgfC, bgrR and bgrS was significantly downregulated in △cel‐EIIB strain (p < 0.05). In conclusion, cel‐EIIB protein‐mediated cel‐PTS not only contributes to biofilm formation and virulence regulation, but also plays an important role in the invasion and colonization of S. agalactiae.     

Dietary supplement of fructooligosaccharides and Bacillus subtilis enhances the growth rate and disease resistance of the sea cucumber Apostichopus japonicus (Selenka)

The effects of two dietary supplements, entrapped fructooligosaccharide (FOS) and microencapsulated Bacillus subtilis, on the growth and disease resistance of the sea cucumber Apostichopus japonicus were investigated. Sea cucumbers (a total of 810 animals, with initial weight of 3.72 ± 0.16 g each) were fed with basal diet only or basal diet supplemented with three different doses of B. subtilis (0.2, 0.4 and 0.8%), three different doses of FOS (0.4, 0.8 and 1.6%), 0.2% B. subtilis plus 0.4% or 0.8% FOS. Growth and lysozyme activity were measured after 50 days feeding. The results showed that dietary 0.2% and 0.4% B. subtilis alone or 0.2% B. subtilis plus 0.8% FOS significantly increased the specific growth rate (SGR) of sea cucumbers compared with control (P < 0.05). The addition of B. subtilis with or without FOS had no significant effect on lysozyme activity. The animals were also challenged with Vibrio splendidus to assess the level of disease resistance provided by the two dietary supplements. Sea cucumbers fed with 0.8% B. subtilis or 0.2% B. subtilis plus 0.8% FOS exhibited the highest survival (P > 0.05). Under these experimental conditions, dietary B. subtilis and FOS had no synergistic effect on enhancing SGR and disease resistance of sea cucumber.     

Effect of Indian lotus (Nelumbo nucifera Gaertn.) leaf powder on immune status and disease resistance of Nile tilapia

This study assessed the immune response of Nile tilapia (Oreochromis niloticus L.) after feeding on different levels (0.1%, 0.2% and 0.4%) of dietary Indian lotus (Nelumbo nucifera) leaf powder for 45 days. We evaluated both the nonspecific immune response at the end of the feeding period and the resistance to Aeromonas hydrophila. Exposure to Indian lotus resulted in a significant elevation in serum total globulins, serum lysozyme activity, serum killing percentage and the phagocytic activity (p < 0.05). Total serum protein and albumin showed no remarkable variation between tilapia fed on 0.1% Indian lotus and the control group (p > 0.05). In addition, the relative expressions of immune‐related genes, namely interleukin–1β and tumour necrosis factor–α were significantly up‐regulated in tilapia fed on 0.4% Indian lotus as compared to the control group; their expressions were down‐regulated in the other tested groups (p < 0.05). The survival rate of Nile tilapia postchallenge to A. hydrophila reported a significant and dose‐dependent increase in the Indian lotus‐supplemented groups (p < 0.05). Therefore, dietary incorporation of Indian lotus leaves (0.4%, 0.2% and 0.1%) could strengthen the immunity of Nile tilapia and improve its resistance to A. hydrophila infection. Therefore, Indian lotus leaves could serve as potential feed supplements for Nile tilapia.     

Evaluation of carbohydrate‐to‐lipid ratio in diets supplemented with Bacillus subtilis probiotic strain on growth performance,body composition and digestibility in juvenile white seabass (Atractoscion nobilis,Ayres 1860)

This study aimed to evaluate the effect of increasing the carbohydrate to lipid ratio in diets supplemented with Bacillus subtilis in white seabass (Atractoscion nobilis) juveniles on digestibility, growth performance, feed utilization efficiency and tissue composition. Four experimental isoenergetic and isoproteic diets supplemented with B. subtilis were formulated with increasing starch to lipid ratios (g/g) of 10:19; 14:17; 18:15 and 22:13 (diets PD10, PD14, PD18 and PD22 respectively). A negative control diet for B. subtilis supplementation was also formulated to be similar to diet PD10 (diet CD10). Each experimental diet was fed to triplicate groups of 20 fish per tank (IBW: 9.5 g), three times a day, to apparent satiation, for 60 days. Supplementation with B. subtilis, of the low starch‐high lipid diet (10:19), significantly enhanced the overall growth performance, digestibility and feed efficiency. Moreover, the increase in starch to lipid ratio, in supplemented probiotic diets, significantly increased growth performance, feed intake, feed utilization efficiency and protein and energy digestibility, being maximum for the highest dietary starch to lipid level. Lipid content of whole body, muscle and liver and hepatosomatic index significantly increased with the increase in starch to lipid ratio. The results of this study indicate that B. subtilis supplementation significantly enhanced growth performance, digestibility and feed utilization of low starch‐high lipid diet. However, further improvement of growth performance and feed utilization efficiency may be achieved combining B. subtilis supplementation with increased dietary starch to lipid ratio to 22:13.