Panchalika Deachamag

Highly sensitive capacitive biosensor for detecting white spot syndrome virus in shrimp pond water.

Water is one major pathways by which the white spot syndrome virus (WSSV) pathogen enters aquaculture facilities. This paper describes the production and use of a capacitive biosensor for the quantitative detection of as little as 1copy/μl of WSSV in shrimp pond water. A glutathione-S-transferase tag for white spot binding protein (GST-WBP) was immobilized on a gold electrode through a self-assembled monolayer. Binding between WSSV and the immobilized GST-WBP was directly detected by a capacitance measurement. Under optimum conditions, the capacitive biosensor detected WSSV over a wide linear range of between 1 and 1 × 10(5)copies/μl. The system was highly selective for WSSV. One analysis cycle required only 20-25 min of analysis time and 25 min of regeneration time. The capacitive biosensor was applied to analyze WSSV concentration in eight shrimp pond water samples and the results were in good agreement with those obtained by a real time quantitative polymerase chain reaction (real-time PCR) method (P>0.05). The immobilized GST-WBP provided and could be reused for up to 39 analysis cycles for one electrode preparation with a relative standard deviation (RSD) of 2.4% and a good reproducibility of residual activity (95.8 ± 2.3%). The appealing performance of this biosensor indicated that it had great potential for an accurate very sensitive, quantitative, detection method for WSSV.

WSSV: VP26 binding protein and its biological activity.

White spot syndrome virus (WSSV) is one of the major causes of disease in the shrimp culture industry causing enormous economic losses. In this study, we displayed peptides from a cDNA library obtained from the hemolymph of shrimp infected with WSSV, on the surface of phage and screened for the peptides that interacted with the WSSV. One WSSV binding protein (WBP) gene was found to consist of 171 bp that had no matches in the NCBI database. This WBP was shown to bind to the VP26 protein of the WSSV by Western blotting. In addition, WBP reduced the binding of WSSV to shrimp haemocytes from 2.0 × 10(7)copies in the control to 6.0 × 10(2) after treatment with 80 μg of WBP. The survival rate of shrimp after WSSV were mixed with WBP at 80 μg, was 89% and the binding of WBP remained unchanged for at least 24h. Therefore, the results indicate that the WBP can bind to VP26 and inhibit the invasion of WSSV into host cells. This finding may introduce another future way to try to fight this disease in shrimp culture.

Stimulating the immune response of Litopenaeus vannamei using the phagocytosis activating protein (PAP) gene.

High mortality in the shrimp farming industry is caused by several pathogens such as white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio harveyi (V. harveyi). A PAP (Phagocytosis activating protein) gene able to activate phagocytosis of shrimp hemocytes was cloned into the eukaryotic expression vector phMGFP. In vitro expression was confirmed by transfection of PAP-phMGFP into CHO (Chinese Hamster Ovary) cells and the expression of the Green Fluorescent Protein (GFP) was observed. In order to activate the phagocytic activity of shrimp, 20, 40 and 80 μg/shrimp of this PAP-phMGFP vector were injected into Litopenaeus vannamei muscle. After challenged with WSSV, 40 μg/shrimp produced the highest relative percent survival (77.78 RPS). Analysis for the expression of the GFP gene in various tissues showed the expression mostly in the hemolymph of the immunized shrimp. The expression level of PAP and proPO (Prophenoloxidase) gene were highest at 7 days after immunization. This agreed with the efficiency of protection against WSSV that also occurred 7 days after immunization with the highest RPS of 86.61%. However there was no protection 30 days after immunization. Hemocytes of shrimp injected with PAP-phMGFP had 1.9 folds and 3 folds higher percentage phagocytosis and phagocytic index than the shrimp injected with PBS. Accordingly, copies of WSSV reduced in the PAP-phMGFP injected shrimp. In addition, PAP-phMGFP also protected shrimp against several pathogens: WSSV, YHV and V. harveyi, with RPS values of 86.61%, 63.34% and 50% respectively. This finding shows that the immune cellular defense mechanisms in shrimp against pathogens can be activated by injection of PAP-phMGFP and could indicate possible useful ways to begin to control this process.

Ovarian Transcriptome Analysis of Vitellogenic and Non-Vitellogenic Female Banana Shrimp (Fenneropenaeus merguiensis)

The banana shrimp (Fenneropenaeus merguiensis) is one of the most commercially important penaeid species in the world. Its numbers are declining in the wild, leading to a loss of broodstock for farmers of the shrimp and a need for more successful breeding programs. However, the molecular mechanism of the genes involved in this shrimp’s ovarian maturation is still unclear. Consequently, we compared transcriptomic profiles of ovarian tissue from females in both the vitellogenic stage and the non-vitellogenic stage. Using RNA-Seq technology to prepare the transcriptome libraries, a total of 12,187,412 and 11,694,326 sequencing reads were acquired from the non-vitellogenic and vitellogenic stages respectively. The analysis of the differentially expressed genes identified 1,025 which were significantly differentially expressed between the two stages, of which 694 were up-regulated and 331 down-regulated. Four genes putatively involved in the ovarian maturation pathway were chosen for validation by quantitative real-time PCR (RT-qPCR). The data from this study provided information about gene expression in ovarian tissue of the banana shrimp which could be useful for a better understanding of the regulation of this species’ reproductive cycle.

Development of an immuno-based colorimetric assay for white spot syndrome virus.

White spot syndrome virus (WSSV) is a major cause of infectious disease in cultured shrimp. A fast and reliable method for detecting and monitoring the amount of WSSV during farming would be extremely useful. This work describes a sandwich immunoassay that uses anti‐GST‐VP26, a WSSV‐binding protein (WBP), and modified streptavidin magnesphere paramagnetic particles (SMPPs) to develop the technique. The WBP was immobilized on SMPPs and later bound to different copies of WSSV. The binding was detected using anti‐GST‐VP26 conjugated to alkaline phosphatase. This enzymatic reaction successfully changed the test solution to a concentration‐dependent yellow color that was measured at 405 nm. The sensitivity of this method was between 1.6 × 104 and 1.6 × 107 copies µL−1 of WSSV. In this study, the color for detection and semiquantitative analysis is easily observed and measured and can lead to the development of a test kit for screening WSSV during shrimp farming.

In silico analysis of protein toxin and bacteriocins from Lactobacillus paracasei SD1 genome and available online databases

Lactobacillus paracasei SD1 is a potential probiotic strain due to its ability to survive several conditions in human dental cavities. To ascertain its safety for human use, we therefore performed a comprehensive bioinformatics analysis and characterization of the bacterial protein toxins produced by this strain. We report the complete genome of Lactobacillus paracasei SD1 and its comparison to other Lactobacillus genomes. Additionally, we identify and analyze its protein toxins and antimicrobial proteins using reliable online database resources and establish its phylogenetic relationship with other bacterial genomes. Our investigation suggests that this strain is safe for human use and contains several bacteriocins that confer health benefits to the host. An in silico analysis of protein-protein interactions between the target bacteriocins and the microbial proteins gtfB and luxS of Streptococcus mutans was performed and is discussed here.

Semiquantitative dot‐blot immunogold assay for specific detection of white spot syndrome virus

A dot‐blot immunogold assay (DBIA) was developed to detect white spot syndrome virus (WSSV) using the polyclonal antibody VP26 (anti‐VP26). The anti‐VP26 was immobilized on gold nanoparticles (Ab‐AuNPs), and a nitrocellulose membrane was used as a detection pad. When the target WSSV bound to the Ab‐AuNPs a reddish dot appeared on the surface of the membrane used within 2–5 Min, which could be seen with the naked eye. The test was able to detect WSSV at concentrations as low as 105 copies μL−1 of WSSV. The DBIA developed had good specificity, and the colloidal gold probe can be applied within 2–3 days when stored at 4 °C. For real sample analysis, the DBIA was applied to samples of seawater used for shrimp cultivation without sample preparation. The results indicate that sample 1 showed a positive result, whereas samples 2 and 3 produced negative results. Then, samples 2 and 3 were spiked with WSSV for method validation. To confirm the performance of the DBIA developed, polymerase chain reaction (PCR) was conducted and the PCR results were the same as those found by the DBIA. Therefore, the DBIA developed could be applied for WSSV detection in real water samples.

Antimicrobial activity of engineered shrimp ovarian peritrophin fragments from Fenneropenaeus merguiensis.

Shrimp ovarian peritrophin (SOP), a major protein in jelly layer and cortical rods, plays a role in egg protection after spawning. Previous study, sequence of SOP gene from Fenneropenaeus merguiensis (Fm-SOP) was composed of domain A and domain B. The SOP domain A contains amino acid sequences between 1-80 of Fm-SOP. The domain A had six conserved cysteines which have been found in many antimicrobial peptides. The molecular weight of purified rSOP-A protein was about 9 kDa. The SOP domain B contains amino acid sequences 81-329 of Fm-SOP while SOP-B1 was amino acid sequence 182-275 of Fm-SOP. The molecular weight of purified rHis-SOP-B and rHis-SOP-B1 protein were about 38.5 and 18.0 kDa, respectively. Antimicrobial activities of rSOP-A, rHis-SOP-B and rHis-SOP-B1 protein were investigated by liquid growth inhibition assay. Minimal Inhibition Concentration (MIC) of rSOP-A against Staphylococcus aureus, Escherichia coli, Vibrio harveyi, Candida albicans and Fusarium oxysporum were 35, 280, 280, 570 and 15 µg/mL, respectively. The MIC of rHis-SOP-B against S. aureus, V. harveyi and F. oxysporum were 30, 270 and 500 µg/mL, respectively. And the MIC of rHis-SOP-B1 against S. aureus, V. harveyi and F. oxysporum were 20, 470 and 250 µg/mL, respectively. The rHis-SOP-B and rHis-SOP-B1 (1000 µg/mL) did not show antimicrobial activity against E. coli and C. albicans. Three purified proteins were able to agglutinate V. harveyi in vitro, displayed a chitinase activity and proteinase inhibition. In addition the stability of the proteins was tested and found decrease antimicrobial activity after incubation at 50 °C for 5 h.