Decreased Severity of Acute Hepatopancreatic Necrosis Disease in White Shrimp (Litopenaeus vannamei) by Mixed Culture of Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium.

The objective of this study was to investigate the mixed culture of Bacillus subtilis, B. licheniformis and B. megaterium to control acute hepatopancreatic necrosis disease (AHPND) or EMS (Early Mortality Syndrome) in white shrimp Litopenaeus vannamei as a model. The infected shrimps with Vibrio parahaemolyticus AHPND strain were divided into tanks and different feeding of either B. subtilis, B. licheniformis, B. megaterium or all Bacillus strains. The infected shrimps that were fed with a mixed culture of Bacillus showed significantly highest survival rate and revealed lower percent detection of V. parahaemolyticus AHPND strain by Polymerase Chain Reaction (PCR) (57.14%) with a small amount of viability count in their hepatopancreas. In contrast, the infected shrimps that were fed with each of B. subtilis, B. licheniformis or B. megaterium, revealed the spread of V. parahaemolyticus AHPND strain in all tissue by PCR detection (86.67%-100%) with a large amount of viability count (3.53 - 4.24 × 103 CFU/g). This study indicated that the mixed culture of Bacillus subtilis, B. licheniformis and B. megaterium could control the dissemination of V. parahaemolyticus in shrimps, especially in hepatopancreatic that is the target tissue of AHPND in white shrimp (L. vannamei). The result of this study revealed the efficiency and mechanism of the mixed culture of B. subtilis, B. licheniformis and B. megaterium to control the virulence of AHPND and support the application of this mixed culture in aquaculture of shrimp farms to avoid chemical and antibiotic treatment by using it as a biological control.

First Draft Genome Sequence of Thermophilic Laceyella tengchongensis BKK01, Isolated from Municipal Solid Waste in Thailand.

Laceyella tengchongensis BKK01 is a thermophilic bacterium isolated from municipal solid waste. The genome of L. tengchongensis BKK01 includes a gene putatively encoding gramicidin S synthase. Gramicidin S has antibiotic activity against some bacteria and fungi. The newly sequenced 3.44-Mb draft genome of L. tengchongensis BKK01 will shed some light on the biosynthesis of gramicidin S.

Draft Genome Sequence of Haloferax volcanii SS0101, Isolated from Salt Farms in Samut Sakhon, Thailand.

Haloferax volcanii SS0101 is a halophilic archaeon isolated from salt farms in Thailand. The genome sequence of H. volcanii SS0101 contains a gene encoding capreomycidine synthase, a key enzyme for capreomycidine biosynthesis. This 3.8-Mb draft genome sequence of H. volcanii SS0101 will provide the tools for investigating genes involved in capeomycidine production in haloarchaea.

Expression and characterization of Cu/Zn superoxide dismutase from Wuchereria bancrofti.

The Cu/Zn superoxide dismutase gene from Wuchereria bancrofti (Cu/Zn WbSOD) was isolated by PCR using degeneracy primers. The complete Cu/Zn WbSOD consisted of 1,032 nucleotides containing 4 exons (477 nucleotides) and 3 introns. The molecular phylogenetic analysis of the Cu/Zn WbSOD gene in comparison with those from other organisms revealed that the gene was classified in the same clade to those of filarial Brugia malayi and Brugia pahangi (bootstrap value at 90). The nucleotide and deduced amino acid sequences of Cu/Zn WbSOD exhibited the similarity to those of intracellular Cu/Zn SOD of B. malayi and B. pahangi. The amino acid comparison of Cu/Zn WbSOD to others revealed that the binding sites and active sites were conserved. The expression of this gene yielded 16.366 kDa in size. After Ni-IDA column purification, the enzyme showed specific activity of 8.5 U/mg and 42.1% yield. The enzyme activity was inhibited when 6 mM KCN was added.

Cloning, expression, and characterization of thermotolerant manganese superoxide dismutase from Bacillus sp. MHS47.

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7-8.5, with an optimum pH of 7.5, and at temperatures in the range 30-45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.

Purification and characterization of organic solvent and detergent tolerant lipase from thermotolerant Bacillus sp. RN2.

The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2). The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9-11 and temperature range of 45-60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8) and pNP-laurate (C12) and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

High resolution melting real-time PCR for rapid discrimination between Brugia malayi and Brugia pahangi.

OBJECTIVE: To identify two closely related Brugia malayi and B. pahangi in cat reservoirs by using high resolution melting real-time PCR (HRM real-time PCR). MATERIAL AND METHOD: HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 5 Brugia specimens by using five sets of specific primers for HhaI repetitive region (HR), small heat shock protein (SHP), small subunit ribosomal DNA (18S rDNA), internal transcribed spacer region (ITS), and trans-spliced leading Exon I gene (SLX1). RESULTS: HRM analysis of ITS and SLX clearly generated 2 profiles of B. malayi and B. pahangi while those of HR, 18S rDNA, and SHP could classify B. pahangi. CONCLUSION: HRM is a simple and rapid method for identification of two closely related B. malayi and B. pahangi in which it can detect both parasites within 30 min after real-time PCR detection. This assay is probe-free HRM and reduces a risk of PCR carryover. It does not require multiplex methods and DNA sequencing; therefore, HRM provides a new approach for genetic screening and rapid detection of closely related species in a clinical laboratory.

Molecular genetics analysis for co-infection of Brugia malayi and Brugia pahangi in cat reservoirs based on internal transcribed spacer region 1.

This study described the diagnosis of a mixed infection of Brugia malayi and Brugia pahangi in a single domestic cat using the internal transcribed spacer 1 (ITS1) region. Following polymerase chain reaction amplification of the ITS1 region, the 580 bp amplicon was cloned, and 29 white colonies were randomly selected for DNA sequencing and phylogenetic tree construction. A DNA parsimony tree generated two groups of Brugia spp with one group containing 6 clones corresponding to B. pahangi and the other 23 clones corresponding to B. malayi. This indicated that mixed infection of the two Brugia spp, B. pahangi and B. malayi, had occurred in a single host.

Intraspecies variation of Brugia spp. in cat reservoirs using complete ITS sequences.

The internal transcribed spacer (ITS) region was used to study the intraspecies variation of Brugia spp. in cat reservoirs. Blood specimens from seven naturally infected cats were collected from two different geographical brugian-endemic areas in Thailand. The DNAPAR tree of these Brugia spp. was constructed using a maximum likelihood approach based on ITS nucleotide sequences and was compared to those of Brugia malayi, Brugia pahangi, and Dirofilaria immitis that were previously reported in GenBank. The phylogenetic trees inferred from ITS1, ITS2, and complete ITS sequences indicated that B. malayi and B. pahangi were separated into two clades, and subgroups were generated within each clade. The data revealed that ITS2 sequences were less informative than ITS1 for studying intraspecies variation of Brugia spp. Our results are primary data for intraspecies variation of B. malayi and B. pahangi in cat reservoirs. The information could be applicable for studying the molecular epidemiology and the dynamic nature of the parasites.

Genetic diversity of Trypanosoma evansi in beef cattle based on internal transcribed spacer region.

This study was focused on genetic diversity of Trypanosoma evansi which is a widely distributed haemoflagellate of veterinary importance that infects a variety of larger mammals including horses, mules, camels, buffalo, cattle and deer. The genetic diversity of T. evansi of beef cattle LAM19 was accomplished by using phylogenetic analysis based on internal transcribed spacer region (ITS). Blood sample was collected from a naturally infected beef cattle LAM 19 and parasitemia was raised by mouse inoculation. The parasites were collected and isolated by using DE 52 DEAE cellulose anion exchange column prior to DNA extraction. Upon PCR amplification of ITS region, the product of 1300bp in size was obtained. The ITS nucleotide sequences were analyzed and revealed that it could demonstrate the genetic diversity of T. evansi of beef cattle LAM19. Based on the ITS tree, beef cattle LAM 19 T. evansi were categorized into two main groups where the genetic diversity occurred within Group 1. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.