CpG ODN mimicking CpG rich region of myxosporean Myxobolus supamattayai stimulates innate immunity in Asian sea bass (Lates calcarifer) and defense against Streptococcus iniae.

Oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine CpG dinucleotides within specific sequence contexts (CpG motifs) have been reported as pathogen-associated molecular patterns (PAMPs). Its immunostimulatory effects have been demonstrated in diverse vertebrate models. CpG ODN is typically found in bacterial or viral genome and recognized by a non-self recognition receptor Toll-like receptor9 (TLR9). Here, a new CpG ODN 1013 which mimics sequence of SSU rDNA of early eukaryotic organism myxosporidia, Myxobolus supamattayai, was employed to stimulate the immune responses of Asian sea bass Lates calcarifer. Its immunostimulant potentiality was comparatively compared with that of CpG ODN 1668, a widely used as functional immunostimulant. Both unmethylated CpG ODNs with some modified phosphorothioated positions were intraperitoneally injection (5 µg/fish). Hematological examination, immunological assays and immune-related genes expression were evaluated 12 h, 1, 3 and 5 d after post CpG ODN challenge. The immunosimulatory effect of these CpG ODNs on fish immunity to protect the bacterial pathogen Streptococcus iniae was also determined. The results demonstrated that these two CpG ODNs could induce immune responses in Asian sea bass including the significant (P < 0.05) increase level of WBC, peroxidase activity and oxidative radicals in head kidney (HK) leukocyte, serum innate immune parameters and up-regulation of four immune responsive genes compared with the control group. Most of immune responses induced by ODN 1668 were strong within 1 d but lesser extended while ODN 1013 prolonged the stimulatory effects during the whole experimental period. After challenge with S. iniae, the survival proportion in ODN 1013-treated fish was apparently higher than that treated with ODN 1668 and PBS, respectively. The results together suggested that CpG ODN 1013 enhanced innate immune responses, including humoral and cellular responses, through TLR9 mediated signaling pathway which is mainly contribute to the protective immunity in Asian sea bass against S. iniae infection. These findings can lead to a new approach in immunostimulant development by using the novel CpG ODN originating from the parasite M. supamattayai, besides those from bacterial and viral genomes, for disease control in fish host.

Molecular cloning and characterization of a novel bi-functional α-amylase/subtilisin inhibitor from Hevea brasiliensis.

A novel cDNA encoding a bi-functional α-amylase/subtilisin inhibitor (HbASI) was isolated from rubber (Hevea brasiliensis) leaves cultivar RRIM600. The HbASI had strong homology with the soybean trypsin inhibitor (Kunitz) family of protease inhibitors. Its putative amino acid sequence was similar to that of the α-amylase/subtilisin inhibitor from Ricinus communis (72% identity). Genomic sequencing indicated that the HbASI gene contained no introns. The messenger RNA of HbASI was detected in leaf, hypocotyl and root. The recombinant HbASI expressed extracellularly in Pichia pastoris exhibited inhibitory activity against α-amylase from Aspergillus oryzae, trypsin and subtilisin A. The HbASI gene was induced in the rubber leaves infected with a rubber tree pathogen, Phytophthora palmivora. It was also enhanced by salicylic acid (SA) treatment and mechanical wounding. In addition, the biological activity of the HbASI protein involving in the plant defence responses was also investigated. The HbASI at a concentration of 0.16 mg mL(-1) could inhibit the mycelium growth of P. palmivora. These data suggested that the HbASI protein might play a crucial role in defence against pathogen of rubber trees.

Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization.

Peroxidases are oxidoreductase enzymes produced by most organisms. In this study, a peroxidase was purified from Hevea brasiliensis cell suspension by using anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) and preparative SDS-PAGE. The obtained enzyme appeared as a single band on SDS-PAGE with molecular mass of 70 kDa. Surprisingly, this purified peroxidase also had polyphenol oxidase activity. However, the biochemical characteristics were only studied in term of peroxidase because similar experiments in term of polyphenol oxidase have been reported in our pervious publication. The optimal pH of the purified peroxidase was 5.0 and its activity was retained at pH values between 5.0-10.0. The enzyme was heat stable over a wide range of temperatures (0-60°C), and less than 50% of its activity was lost at 70°C after incubation for 30 min. The enzyme was completely inhibited by ß-mercaptoethanol and strongly inhibited by NaN3; in addition, its properties indicated that it was a heme containing glycoprotein. This peroxidase could decolorize many dyes; aniline blue, bromocresol purple, brilliant green, crystal violet, fuchsin, malachite green, methyl green, methyl violet and water blue. The stability against high temperature and extreme pH supported that the enzyme could be a potential peroxidase source for special industrial applications.

Defense-related polyphenol oxidase from Hevea brasiliensis cell suspension: purification and characterization.

Polyphenol oxidase (PPO) was examined from the extract of leaf, seed, and cell suspension of Hevea brasiliensis, a rubber plant. The defense-related isozyme from Hevea cell suspension induced by culture filtrate of Phytophthora palmivora or by agitation stress was isolated through anion exchange and affinity chromatography, respectively. A 104-purification fold, migrated as a single band of 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PPO, was obtained after further purified by the preparative gel electrophoresis. Based on reaction with catechol and dopamine but not with p-cresol and guaiacol, it is a diphenol-type PPO. The values of V(max)/K(m) ratio indicated that catechol was the most specific substrate. The optimal activity of the purified PPO was observed at pH 6.0. The PPO activity was retained at pH 4.0-10.0 and temperature 10-60 °C. The inhibitors which completely inhibited the activity were ascorbic acid, dithiothreitol, and ß-mercaptoethanol while sodium azide was a poor inhibitor. The PPO obtained from Hevea cell suspension possesses high specific activity and is stable at wide range of pH and temperature. It is therefore suitable for extreme condition uses and may lead to an alternative source of PPO in various industrial applications.

A new host record of Sphaerospora epinepheli (Myxosporea: Bivalvulida) occurring on orange-spotted grouper Epinephelus coioides from Thailand: epidemiology, histopathology and phylogenetic position.

In 1991, the first record of Sphaerospora epinepheli was described as a kidney parasite of wild and cultured malabar grouper, Epinephelus malabaricus, along coastlines of Thailand, the Gulf of Thailand and the Andaman Sea. However, the present study detected high infection of this parasite in kidney renal tubes of orange spotted grouper, Epinephelus coioides, collected from Andaman Sea. The highest infection rate of 36.82% was observed during the rainy season in 2009 in Phang-Nga Bay, in the north of Andaman Sea, which is an important grouper production site in Thailand. The biological and histopathological data of the parasite in this new host record are presented. Species classification is described based on morphological data of mature spore and molecular analysis of myxosporean 18S rDNA phylogeny including that of S. epinepheli which infected E. malabaricus. The genetic position of this parasite found in two host species was also studied. The phylogenetic tree analysis of small-subunit rDNA sequences of S. epinepheli from both infected hosts was constructed using two algorithms, maximum likelihood (ML) and Bayesian inference (BI). They were placed in the clustered basal sphaerosporid clade that contain four long SSU rDNA sphaerosporid species including Sphaerospora truttae, Sphaerospora elegans, Sphaerospora ranae, Sphaerospora fugu and Bipteria formosa with strong bootstrap supports. Histopathologically, renal intratubular myxosporean spores were associated with tubulonephosis, tubular necrosis, chronic interstitial nephritis and mimic membranoproliferative glomerulonephritis. This myxosporean parasite appears to be a significant pathogen on the basis of pathological changes in the renal tubules and is highly distributed in orange-spotted grouper.