Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shr... more Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shrimp within 2-3 days after the first appearance of gross signs of disease in rearing ponds. Current detection systems for YHV are based on molecular and immunological techniques. Immunological detection methods require continual production of relatively large, complex proteins using living animals or hybridoma cells. An alternative possibility is to use the phage display technique to find short peptides that can bind strongly with YHV particles and replace antibodies in such tests. Once identified, these peptides could also be tested for efficacy in blocking YHV infection. YHV was purified and then immobilized on microtiter plates for 4 rounds of biopanning against a pool of phage displayed peptide variants. From 89 sequentially generated phage pools, 13 variants were selected for strong binding with YHV by ELISA assay. DNA sequencing led to the deduced amino acid sequences LNAKSRN, KSKKS...
Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shr... more Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shrimp within 2-3 days after the first appearance of gross signs of disease in rearing ponds. Current detection systems for YHV are based on molecular and immunological techniques. Immunological detection methods require continual production of relatively large, complex proteins using living animals or hybridoma
Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been exten... more Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral administration.
We sequenced four Vibrio parahaemolyticus strains, three of which caused serious acute hepatopanc... more We sequenced four Vibrio parahaemolyticus strains, three of which caused serious acute hepatopancreatic necrosis disease. Sequence analysis of the virulent strains revealed not only genes related to cholera toxin and the type IV pilus/type IV secretion system but also a unique, previously unreported, large extrachromosomal plasmid that encodes a homolog to the insecticidal Photorhabdus insect-related binary toxin PirAB.
In our research efforts to reduce the impact of white spot syndrome virus (WSSV) disease outbreak... more In our research efforts to reduce the impact of white spot syndrome virus (WSSV) disease outbreaks in shrimp aquaculture, we studied the effect of β-glucan administration to activate the prophenoloxidase (proPO) enzymatic cascade prior to WSSV challenge. Injection of a single dose of β-glucan (5 μg/g) prior to WSSV challenge resulted in activation of the proPO system and reduced shrimp mortality (25-50%) when compared to controls (100%). By contrast, no significant reduction was observed using yellow head virus (YHV) in a similar protocol. We subsequently hypothesized that administration of a second dose of β-glucan after WSSV challenge might reduce shrimp mortality further. Surprisingly, the opposite occurred, and mortality of the WSSV-infected shrimp increased to 100% after the second β-glucan dose. Both immunofluorescence and RT-PCR assays revealed low WSSV levels in hemocytes of shrimp collected after the second dose of β-glucan administration, suggesting that the cause of increased mortality was unlikely to be increased WSSV replication. We found from measured phenoloxidase acitivity (PO) and H2O2 production that the higher mortality may have resulted from a combination of WSSV infection plus over-production of reactive oxygen species (ROS) stimulated by two doses of β-glucan. Thus, caution may be prudent in continuous or prolonged activation of the shrimp immune system by β-glucan administration lest it exacerbate shrimp mortality in the event of WSSV infection.
White spot syndrome virus (WSSV) is one of the most serious pathogens in shrimp aquaculture. A sh... more White spot syndrome virus (WSSV) is one of the most serious pathogens in shrimp aquaculture. A shrimp WSSV-binding protein called PmRab7 has been isolated and characterized. Since injection of bacterial expressed-rPmRab7 could reduce shrimp mortality caused by WSSV from approximately 95% to 15% mortality, there was potential for its use in protection against WSSV in shrimp aquaculture. To test the feasibility of this, the Pichia pastoris yeast expression system was used for production of rPmRab7 since its expression system has eukaryote post-translational modification capability and since P. pastoris is widely accepted for use in human food or animal feed. Moreover, β-1,3-glucan, a major cell wall component of yeast, has been reported to act as an immunostimulant in shrimp. The recombinant protein was produced intracellularly and the resulting whole yeast cells were lyophilized and stored for supplementation in shrimp feed. The yield of rPmRab7 was 20-30 mg/l of culture medium or 2.67 mg/g yeast dry weight, which was 2-3 times higher than the yield obtained from an Escherichia coli expression system. A two-copy gene expression system was developed to enhance rPmRab7 expression using expression vector pAO815 containing a two-copy PmRab7 expression cassette constructed by site-directed mutagenesis of the PmRab7 gene and two-step overlap, extension PCR. This improved the yield of rPmRab7 2-3 times (40-60 mg/l of culture medium). ELISA was developed to show that the expressed rPmRab7 had WSSV-binding activity. Although some loss of rPmRab7 was found after lyophilization of the yeast cells, projected cost calculations indicated that this production level would make it feasible to use rPmRab7 in shrimp feed for protection against WSSV.
ABSTRACT The Thai Department of Fisheries (DOF), 2013 estimated that outbreaks of acute early mor... more ABSTRACT The Thai Department of Fisheries (DOF), 2013 estimated that outbreaks of acute early mortality (often called early mortality syndrome or EMS) in cultivated shrimp were responsible for a 33% drop in shrimp production during the first quarter of 2013. Similar early mortality in Vietnam was ascribed to specific isolates of Vibrio parahaemolyticus that caused acute hepatopancreatic necrosis disease (AHPND) but the status of EMS/AHPND in Thailand was unclear. Here we describe the isolation and characterization of bacteria isolated from the hepatopancreas (HP) of shrimp collected from an early mortality outbreak farm in Thailand. Four independent bacterial isolates were identified as V. parahaemolyticus by BLAST analysis and by gene-specific marker detection of a lecithin dependent hemolysin (LDH) considered to be specific for the species. Immersion challenges with 3 of these and a reference isolate, obtained from China in 2010, using a previously published laboratory infection model caused very high mortality accompanied by characteristic AHPND histopathology in the shrimp HP. Tests with one of these isolates (5HP) revealed that rate of mortality was dose dependent. Using the same challenge protocol, the 4th isolate (2HP) also caused high mortality, but it was not accompanied by AHPND histopathology. Instead, it caused a different histopathology of the HP including collapsed epithelia and unique vacuolization of embryonic cells (E-cells). These results revealed the possibility of diversity in isolates of V. parahaemolyticus that may cause early mortality in shrimp cultivation ponds. Genomic and episomic DNA of these isolates and isolates of V. parahaemolyticus that cause no disease need to be compared to better understand the molecular basis of bacterial virulence in AHPND.
Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shr... more Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shrimp within 2-3 days after the first appearance of gross signs of disease in rearing ponds. Current detection systems for YHV are based on molecular and immunological techniques. Immunological detection methods require continual production of relatively large, complex proteins using living animals or hybridoma cells. An alternative possibility is to use the phage display technique to find short peptides that can bind strongly with YHV particles and replace antibodies in such tests. Once identified, these peptides could also be tested for efficacy in blocking YHV infection. YHV was purified and then immobilized on microtiter plates for 4 rounds of biopanning against a pool of phage displayed peptide variants. From 89 sequentially generated phage pools, 13 variants were selected for strong binding with YHV by ELISA assay. DNA sequencing led to the deduced amino acid sequences LNAKSRN, KSKKS...
Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shr... more Yellow head virus (YHV) causes a serious disease that can result in high mortality of penaeid shrimp within 2-3 days after the first appearance of gross signs of disease in rearing ponds. Current detection systems for YHV are based on molecular and immunological techniques. Immunological detection methods require continual production of relatively large, complex proteins using living animals or hybridoma
Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been exten... more Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral administration.
We sequenced four Vibrio parahaemolyticus strains, three of which caused serious acute hepatopanc... more We sequenced four Vibrio parahaemolyticus strains, three of which caused serious acute hepatopancreatic necrosis disease. Sequence analysis of the virulent strains revealed not only genes related to cholera toxin and the type IV pilus/type IV secretion system but also a unique, previously unreported, large extrachromosomal plasmid that encodes a homolog to the insecticidal Photorhabdus insect-related binary toxin PirAB.
In our research efforts to reduce the impact of white spot syndrome virus (WSSV) disease outbreak... more In our research efforts to reduce the impact of white spot syndrome virus (WSSV) disease outbreaks in shrimp aquaculture, we studied the effect of β-glucan administration to activate the prophenoloxidase (proPO) enzymatic cascade prior to WSSV challenge. Injection of a single dose of β-glucan (5 μg/g) prior to WSSV challenge resulted in activation of the proPO system and reduced shrimp mortality (25-50%) when compared to controls (100%). By contrast, no significant reduction was observed using yellow head virus (YHV) in a similar protocol. We subsequently hypothesized that administration of a second dose of β-glucan after WSSV challenge might reduce shrimp mortality further. Surprisingly, the opposite occurred, and mortality of the WSSV-infected shrimp increased to 100% after the second β-glucan dose. Both immunofluorescence and RT-PCR assays revealed low WSSV levels in hemocytes of shrimp collected after the second dose of β-glucan administration, suggesting that the cause of increased mortality was unlikely to be increased WSSV replication. We found from measured phenoloxidase acitivity (PO) and H2O2 production that the higher mortality may have resulted from a combination of WSSV infection plus over-production of reactive oxygen species (ROS) stimulated by two doses of β-glucan. Thus, caution may be prudent in continuous or prolonged activation of the shrimp immune system by β-glucan administration lest it exacerbate shrimp mortality in the event of WSSV infection.
White spot syndrome virus (WSSV) is one of the most serious pathogens in shrimp aquaculture. A sh... more White spot syndrome virus (WSSV) is one of the most serious pathogens in shrimp aquaculture. A shrimp WSSV-binding protein called PmRab7 has been isolated and characterized. Since injection of bacterial expressed-rPmRab7 could reduce shrimp mortality caused by WSSV from approximately 95% to 15% mortality, there was potential for its use in protection against WSSV in shrimp aquaculture. To test the feasibility of this, the Pichia pastoris yeast expression system was used for production of rPmRab7 since its expression system has eukaryote post-translational modification capability and since P. pastoris is widely accepted for use in human food or animal feed. Moreover, β-1,3-glucan, a major cell wall component of yeast, has been reported to act as an immunostimulant in shrimp. The recombinant protein was produced intracellularly and the resulting whole yeast cells were lyophilized and stored for supplementation in shrimp feed. The yield of rPmRab7 was 20-30 mg/l of culture medium or 2.67 mg/g yeast dry weight, which was 2-3 times higher than the yield obtained from an Escherichia coli expression system. A two-copy gene expression system was developed to enhance rPmRab7 expression using expression vector pAO815 containing a two-copy PmRab7 expression cassette constructed by site-directed mutagenesis of the PmRab7 gene and two-step overlap, extension PCR. This improved the yield of rPmRab7 2-3 times (40-60 mg/l of culture medium). ELISA was developed to show that the expressed rPmRab7 had WSSV-binding activity. Although some loss of rPmRab7 was found after lyophilization of the yeast cells, projected cost calculations indicated that this production level would make it feasible to use rPmRab7 in shrimp feed for protection against WSSV.
ABSTRACT The Thai Department of Fisheries (DOF), 2013 estimated that outbreaks of acute early mor... more ABSTRACT The Thai Department of Fisheries (DOF), 2013 estimated that outbreaks of acute early mortality (often called early mortality syndrome or EMS) in cultivated shrimp were responsible for a 33% drop in shrimp production during the first quarter of 2013. Similar early mortality in Vietnam was ascribed to specific isolates of Vibrio parahaemolyticus that caused acute hepatopancreatic necrosis disease (AHPND) but the status of EMS/AHPND in Thailand was unclear. Here we describe the isolation and characterization of bacteria isolated from the hepatopancreas (HP) of shrimp collected from an early mortality outbreak farm in Thailand. Four independent bacterial isolates were identified as V. parahaemolyticus by BLAST analysis and by gene-specific marker detection of a lecithin dependent hemolysin (LDH) considered to be specific for the species. Immersion challenges with 3 of these and a reference isolate, obtained from China in 2010, using a previously published laboratory infection model caused very high mortality accompanied by characteristic AHPND histopathology in the shrimp HP. Tests with one of these isolates (5HP) revealed that rate of mortality was dose dependent. Using the same challenge protocol, the 4th isolate (2HP) also caused high mortality, but it was not accompanied by AHPND histopathology. Instead, it caused a different histopathology of the HP including collapsed epithelia and unique vacuolization of embryonic cells (E-cells). These results revealed the possibility of diversity in isolates of V. parahaemolyticus that may cause early mortality in shrimp cultivation ponds. Genomic and episomic DNA of these isolates and isolates of V. parahaemolyticus that cause no disease need to be compared to better understand the molecular basis of bacterial virulence in AHPND.
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