Metabolic turnover rate, digestive enzyme activities, and bacterial communities in the white shrimp Litopenaeus vannamei under compensatory growth.

The present work aimed to evaluate the effects promoted by a phase of compensatory growth on metabolic turnover rate, digestive enzyme activity, and bacterial biota of the Pacific white shrimp Litopenaeus vannamei kept under different feeding regimes. Three treatments were evaluated as follows: 70% feed restriction during 3 (T3) and 6 (T6) days, followed by a period of feeding to satiety, and a control treatment without restriction periods. The results showed a full compensatory growth in treatments T3 and T6 by day 35 of the bioassay. A significant increase in trypsin and lipase (T6) activities was observed during compensatory growth, whereas specific amylase activity was significantly lower in treatment T6 compared to T3 but not significantly different from the control group. To determine the metabolic turnover rate of nitrogen in muscle tissue, an analysis of nitrogen isotope values (δ15N) at natural abundance levels was performed. At the end of the experimental period, shrimp under feed restriction had lower metabolic turnover rates and longer nitrogen residence times (t 50) in muscle tissue, as compared to individuals in the control treatment. Regarding the changes in the bacterial communities in shrimp gut, no significant differences were observed at the phylum level, with Proteobacteria being the most abundant bacteria, followed by Actinobacteria. At family taxa level, Rhodobacteraceae presented the highest relative abundance in all treatments, whereas a decrease in Vibrionaceae was observed in treatments T3 and T6 when compared to control shrimps during compensatory growth. At the genus level, a decrease in Celeribacter, Catenococcus, and Epibacterium, and an increase in Ruegeria and Shimia, were identified in shrimp subjected to feed restriction when compared to control organisms during compensatory growth (day 14). At the end of the experimental period, the evaluated parameters showed similar results as those observed in the control treatment, suggesting a normalization of the metabolism and the physiological state. The present findings contribute to a better understanding on the physiological effects produced during compensatory growth in shrimp, which in turn could assist in the development of improved feeding strategies in benefit of the aquaculture industry.

Use of seaweed Ulva lactuca for water bioremediation and as feed additive for white shrimp Litopenaeus vannamei.

Two experimental feeding trials were conducted during four weeks to evaluate the use of Ulva lactuca in shrimp culture: (1) for wastewater bioremediation, and (2) using different inclusion levels of U. lactuca meal in shrimp feed. In feeding trial 1, shrimp reared under seaweed U. lactuca water exchange in a re-circulation system (SWE) resulted in similar growth and feed utilization as shrimp reared with clean water exchange (CWE). Shrimp under no water exchange (NWE) resulted in significant lower growth and higher feed conversion rate (FCR) compared to the other treatments (p < 0.05). Nitrogen compounds and phosphate in water from SWE and CWE treatments did not present significant differences during the experimental trial (p > 0.05). In feeding trial 2, U. lactuca biomass produced by wastewater bioremediation in SWE treatment were dried and ground to formulate diets containing 0, 1, 2, and 3% U. lactuca meal (0UL, 1UL, 2UL, and 3UL). Shrimp fed the 3 UL diet resulted in a significant (p < 0.05) improvement of growth and FCR, and enhanced whole shrimp lipid and carotenoid content by 30 and 60%, respectively, compared to control diet. Seaweed U. lactuca is suggested as a desirable species for wastewater bioremediation in integrated aquaculture systems, and its meal as a good feed additive for farmed shrimp.

Innocuity and anti-Newcastle-virus-activity of Cladosiphon okamuranus fucoidan in chicken embryos.

This study evaluated the potential toxicity and antiviral activity of fucoidan from Cladosiphon okamuranus against Newcastle disease virus (NDV), one of the most serious threats to the poultry industry in the world. Toxicity was assayed on chicken embryo fibroblast (CEF) secondary cultures at concentrations ranging from 0.1 to 1500 µg per mL culture medium, assessing the cell viability by the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, and on 9-day-old embryonated chicken eggs by inoculation of 2 to 500 µg doses in the allantoic cavity, assessing the embryos morphology and liver histology. At 48 h post-inoculation, viability of CEF exposed to concentrations up to 10 µg/mL was not significantly affected, and the 50% cytotoxic concentration was estimated as of 1062 µg/mL; after exposure in ovo, some chick embryos showed liver steatosis when treated with fucoidan doses over 20 µg per egg (15 to 28% at 200 µg, 27 to 56% at 500 µg), but no change was detected in their size or aspect. Antiviral activity was tested by treating 9-day-old embryos via the allantoic route with 0.25 to 16 µg fucoidan doses that were applied at different times (-1, 0 and +1 h) relative to the inoculation of 10,000 folds the 50% Tissue Culture Infective Dose (TCID50) of the NDV, La Sota strain. At 72 h post infection, virus titration in the allantoic fluid by hemagglutination assay (HA) showed a considerable and significant inhibition of infectivity for all doses, the best result (a 90% decrease) being obtained in embryos treated with 1 µg fucoidan one hour before infection. Viral RNA semi-quantification in pooled liver and small intestine of embryos that had been treated with 4 and 16 µg fucoidan 1 h before the infection showed reductions of the virus replication by 60 and 99.8%, respectively. Since this high anti-NDV activity in ovo was obtained with quite innocuous doses, fucoidan from C. okamuranus could be a potential low-toxicity antiviral compound to be used in areas exposed to NDV.

In vitro characterization of the antiviral activity of fucoidan from Cladosiphon okamuranus against Newcastle Disease Virus.

BACKGROUND: Newcastle Disease Virus (NDV) causes a serious infectious disease in birds that results in severe losses in the worldwide poultry industry. Despite vaccination, NDV outbreaks have increased the necessity of alternative prevention and control measures. Several recent studies focused on antiviral compounds obtained from natural resources. Many extracts from marine organisms have been isolated and tested for pharmacological purposes, and their antiviral activity has been demonstrated in vitro and in vivo. Fucoidan is a sulfated polysaccharide present in the cell wall matrix of brown algae that has been demonstrated to inhibit certain enveloped viruses with low toxicity. This study evaluated the potential antiviral activity and the mechanism of action of fucoidan from Cladosiphon okamuranus against NDV in the Vero cell line. METHODS: The cytotoxicity of fucoidan was determined by the MTT assay. To study its antiviral activity, fusion and plaque-forming unit (PFU) inhibition assays were conducted. The mechanism of action was determined by time of addition, fusion inhibition, and penetration assays. The NDV vaccine strain (La Sota) was used in the fusion inhibition assays. PFU and Western blot experiments were performed using a wild-type lentogenic NDV strain. RESULTS: Fucoidan exhibited antiviral activity against NDV La Sota, with an obtained IS50 >2000. In time of addition studies, we observed viral inhibition in the early stages of infection (0-60 min post-infection). The inhibition of viral penetration experiments with a wild-type NDV strain supported this result, as these experiments demonstrated a 48% decrease in viral infection as well as reduced HN protein expression. Ribavirin, which was used as an antiviral control, exhibited lower antiviral activity than fucoidan and high toxicity at active doses. In the fusion assays, the number of syncytia was significantly reduced (70% inhibition) when fucoidan was added before cleavage of the fusion protein, perhaps indicating a specific interaction between fucoidan and the F0 protein. CONCLUSION: The results of this study suggest that fucoidan from C. okamuranus represents a potential low-toxicity antiviral compound for the poultry industry, and our findings provide a better understanding of the mode of action of sulfated polysaccharides.

Antiviral therapy: inhibition of Hepatitis C Virus expression by RNA interference directed against the NS5B region of the viral genome.

BACKGROUND: Hepatitis C virus (HCV) is a major public health problem with 170 million chronically infected people throughout the world. Currently, the only treatment available consists of a combination of pegylated interferon (INF-alpha) and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi) represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV gene expression in short-term cell culture. Our aim was to determine the effect of RNAi directed against the NS5B-HCV region on HCV expression in a human hepatoma cell line that expresses HCV-subgenomic replicon (Huh7 HCV replicon cells). METHODS: We transfected Huh7 HCV replicon cells with different concentrations of RNAi (100-200 nM) targeting the NS5B region of the viral genome. 2-6 days post-transfection HCV-RNA was quantified by semiquantitative and real-time RT-PCR, and HCV NS5B protein levels were assayed by western blot. Cell viability was also quantified by MTT assay. RESULTS: Our results indicate that the NS5B-siRNAs used in this study can specifically inhibit HCV-RNA replication and protein expression (more than 90%) compared to control cells. CONCLUSIONS: Synthetic siRNA against NS5BHCV inhibited HCV replication and viral proteins levels and thereby becomes a powerful strategy to combat hepatitis C virus.