Sasimanas Unajak

Draft Genome Sequences of Six Strains of Vibrio parahaemolyticus Isolated from Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Disease Shrimp in Thailand

ABSTRACT Some strains of Vibrio parahaemolyticus cause acute hepatopancreatic necrosis disease (AHPND) in shrimp. We sequenced 3 AHPND and 3 non-AHPND strains and found that all of them lacked the pathogenicity island relevant to human infection. A unique sequence encoding a type IV pilus/type IV secretion system was found in 3 AHPND strains.

Microarray Analyses of Shrimp Immune Responses

Shrimp aquaculture is one of the major food-producing industries in the world. However, it is being impacted by several problems including diseases, antibiotic use, and environmental factors. The extent of the effects of these problems in the immune system of the shrimp at the molecular level is just beginning to be understood. Here, we review the gene expression profile of shrimp in response to some of these problems using the high-throughput microarray analysis, including white spot syndrome virus, yellow head virus, Vibrio spp., peptidoglycan, oxytetracycline, oxolinic acid, salinity, and temperature.

Effects of the dietary supplementation of mixed probiotic spores of Bacillus amyloliquefaciens 54A, and Bacillus pumilus 47B on growth, innate immunity and stress responses of striped catfish (Pangasianodon hypophthalmus).

ABSTRACT The study used the mixed probiotics of Bacillus amyloliquefaciens 54A and B. pumilus 47B isolated from striped catfish (Pangasianodon hypophthalmus) intestine aiming to stimulate growth performance, innate immunity, stress tolerance of striped catfish. The average weight gain (AWG), specific growth rate (SGR), and feed conversion ratio (FCR) were analyzed after fish were fed the mixture of probiotics (B. amyloliquefaciens 54A and B. pumilus 47B) at concentrations of 1 × 108, 3 × 108, and 5 × 108 CFU g−1 feed for 90 days. Immunity parameters, survival rate of fish challenged with Edwardsiella ictaluri and ammonia tolerance were also investigated. The amounts of B. amyloliquefaciens and B. pumilus were counted and identified by specific primer pairs of Ba1‐F/Ba1‐R, and 16‐F/Bpu‐R to confirm the presence of probiotics in fish intestine. The AWG (476.6 ± 7.81 g fish−1) of fish fed probiotics at 5 × 108 CFU g−1 was significant higher than the control (390 ± 25.7 g fish−1) after 90 days of feeding, but there was no significant (P > 0.05) effect of probiotics on FCR and SGR. Fish fed diet containing probiotics at 5 × 108 CFU g−1 also expressed resistance to E. ictaluri infection and higher immune parameters such as phagocytic activity, respiratory bursts, and lysozyme activity than the control. Stress response with ammonia showed significantly lower mortality rate (25%, 20% and 27%) of fish fed probiotics at all three levels of 1, 3 and 5 × 108 CFU g−1 than the fish fed control diet (75%). The study also demonstrated that the probiotics survived in the intestine of striped catfish after 90 days of feeding. Therefore, the dietary supplementation of a mixture of B. amyloliquefaciens and B. pumilus at 5 × 108 CFU g−1 can be used to improve the health and growth rate of striped catfish. HIGHLIGHTSThe spores of B. amyloliquefaciens 54A and B. pumilus 47B were first applied as probiotics in striped catfish.Dietary supplementation with B. amyloliquefaciens 54A and B. pumilus 47B for 90 days enhanced fish growth.Innate immunity of striped catfish were stimulated after feeding B. amyloliquefaciens 54A and B. pumilus 47B for 90 days.The mixture of B. amyloliquefaciens 54A and B. pumilus 47B in the diet enhanced disease resistance and stress tolerance.Colonization of B. amyloliquefaciens 54A and B. pumilus 47B in the intestine of striped catfish was observed.

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification - by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose - was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0-11.0 and an optimal temperature of approximately 55-60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis-Menten constant (Km) and catalytic constant (Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s(-1), respectively. The catalytic efficiency (Kcat/Km) was 238 s(-1) mM(-1).

Full Length Research Paper Identification of species (meat and blood samples) using nested-PCR analysis of mitochondrial DNA

Crocodile meat product is an alternative protein source. Although, crocodile meat is more expensive, its taste is similar to that of chicken and fish. The authentication of commercial meat species is important for consumer’s confidence. In this study, sensitive and specific method multiplex nested-PCR was applied to identify commercial meat species. Dried blood was used as an alternative DNA source for detection. The detection sensitivity was enhanced by primers specifically designed to encompass the mitochondrial Cytochrome b and NADH dehydrogenase 5/6 genes. The specificity and sensitivity of multiplex PCR system were tested. Different lengths of specific nested-PCR products were detected to be 350, 570, 750 and 1000 bp for chicken, pig, cow, and crocodile, respectively. The system allowed detection with as little as 5 nanogram of DNA from either meat or blood sample. Detection sensitivity of individual species was improved, enabling the detection of DNA with as little as 1 picogram. Cross reaction was not detected among the tested species. It was shown that the multiplex-PCR assay enhanced the sensitivity of routine species identification and allowed the use of blood as an alternative DNA source for detection. Key words : Cytochrome b, NADH dehydrogenase, mitochondrial DNA, meat, blood, species identification, nested-PCR, crocodile.

Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene

Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.

The ribophorin I from Penaeus monodon shrimp: cDNA cloning, expression and phylogenetic analysis☆

Ribophorin I, a 67 kDa subunit of the oligosaccharyl transferase complex, is involved in facilitating N-linked glycosylation of polypeptides. We have isolated a full length Penaeus monodon cDNA encoding an insect/mammalian ribophorin I homologue by screening a lymphoid cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction of lymphoid RNA. The cDNA clone of shrimp ribophorin I (PmRibI) consists of 2263 nucleotides encoding 601 amino acid residues. Primary structure analysis of PmRibI indicated that it is a type I transmembrane protein, comprising a cleavable signal sequence of 23 residues at the amino terminus, preceding 434 residues of the luminal domain, 17 residues of the transmembrane domain, and 150 residues of the cytoplasmic domain at the carboxy terminus. The protein has a calculated molecular mass of 67.98 kDa with a pI of 6.05. This putative PmRibI cDNA clone was also expressed as PmRibI-6His in Sf9 cells. The recombinant PmRibI has an apparent molecular weight of 70 kDa, similar to the MW calculated from the deduced cDNA sequence. The inferred protein sequence of PmRibI has 52% identity with that of Strongylocentrotus purpuratus, 49% identity with that of Danio rerio, and 47% identity with mammalian ribophorin I. Phylogenetic analysis showed that PmRibI is most closely related to the echinoderm ribophorin I. The expression of the ribophorin I gene is tissue specific, with its mRNA highly abundant in hemocytes, gill, lymphoid organ and hepatopancreas.

Molecular serotyping, virulence gene profiling and pathogenicity of Streptococcus agalactiae isolated from tilapia farms in Thailand by multiplex PCR

This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems.

Molecular characterization of Galectin-8 from Nile tilapia (Oreochromis niloticus Linn.) and its response to bacterial infection.

Galectins belong to the family of galactoside-binding proteins and play a major role in the immune and inflammatory responses of vertebrates and invertebrates. The galectin family is divided into three subtypes based on molecular structure; prototypes, chimera types, and tandem-repeated types. We isolated and characterized the cDNA of galectin-8 (OnGal-8) in Nile tilapia (Oreochromis niloticus). OnGal-8 consisted of a 966 bp open reading frame (ORF) that encoded a 321 amino acid protein (43.47 kDa). Homology and phylogenetic tree analysis suggested the protein was clustered with galectin-8s from other animal species and shared at least 56.8% identity with salmon galectin-8. Structurally, the amino acid sequence included two distinct N- and C- terminus carbohydrate recognition domains (CRDs) of 135 and 133 amino acids, respectively, that were connected by a 39 amino acid polypeptide linker. The N- and C-CRDs contained two conserved WG-E-I and WG-E-T motifs, suggesting they have an important role in mediating the specific interactions between OnGal-8 and saccharide moieties such as β-galactoside. The structure of OnGal-8 was characterized by a two-fold symmetric pattern of 10-and 12-stranded antiparallel ß-sheets of both N- and C-CRDs, and the peptide linker presumably formed a random coil similar to the characteristic tandem-repeat type galectin. The expression of OnGal-8 in healthy fish was highest in the skin, intestine, and brain. Experimental challenge of Nile tilapia with S. agalactiae resulted in significant up-regulation of OnGal-8in the spleen after 5 d. Our results suggest that OnGal-8 is involved in the immune response to bacterial infection.

An active recombinant cocoonase from the silkworm Bombyx mori: bleaching, degumming and sericin degrading activities.

BACKGROUND Cocoonase is a serine protease produced by silk moths and used for softening the cocoons so that they can escape. Degumming is one of the important steps in silk processing. This research aimed to produce an active recombinant Bombyx mori cocoonase (BmCoc) for the silk degumming process. RESULTS A recombinant BmCoc was successfully expressed in a Pichia pastoris system. The purified enzyme showed specific activity of 227 U mg(-1) protein, 2.4-fold purification, 95% yield and a molecular weight of 26 kDa. The enzyme exhibited optimal temperature at 40 °C and optimal pH at 8, and showed thermal stability at 25-45 °C and pH stability at 5-9. The recombinant enzyme exhibited sericin degumming ability and color bleaching characteristics, and did not affect the fibroin fiber. The enzyme also degraded sericin substrate with a product size about 30-70 kDa. CONCLUSION In this study, we successfully produced the active recombinant BmCoc in P. pastoris with promising functions for the Thai silk degumming process, which includes degumming, sericin degrading and color bleaching activities. Our data clearly indicated that the recombinant enzyme had proteolytic activity on sericin but not on fibroin proteins. The recombinant BmCoc has proven to be suitable for numerous applications in the silk industry.