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Conventional bacterial genome annotation provides information about coding sequences but ignores untranslated regions and operons. However, untranslated regions contain important regulatory elements as well as targets for many regulatory factors, such as small RNAs. Operon maps are also essential for functional gene analysis. In the last decade, considerable progress has been made in the study of bacterial transcriptomes through transcriptome sequencing (RNA-seq). Given the compact nature of bacterial genomes, many challenges still cannot be resolved through short reads generated using classical RNA-seq because of fragmentation and loss of the full-length information. Direct RNA sequencing is a technology that sequences the native RNA directly without information loss or bias. Here, we employed direct RNA sequencing to annotate the Vibrio parahaemolyticus transcriptome with its full features, including transcription start sites (TSSs), transcription termination sites, and operon maps. A total of 4,103 TSSs were identified. In comparison to short-read sequencing, full-length information provided a deeper view of TSS classification, showing that most internal and antisense TSSs were actually a result of gene overlap. Sequencing the transcriptome of V. parahaemolyticus grown with bile allowed us to study the landscape of pathogenicity island Vp-PAI. Some genes in this region were reannotated, providing more accurate annotation to increase precision in their characterization. Quantitative detection of operons in V. parahaemolyticus showed high complexity in some operons, shedding light on a greater extent of regulation within the same operon. Our study using direct RNA sequencing provides a quantitative and high-resolution landscape of the V. parahaemolyticus transcriptome. IMPORTANCE Vibrio parahaemolyticus is a halophilic bacterium found in the marine environment. Outbreaks of gastroenteritis resulting from seafood poisoning by these pathogens have risen over the past 2 decades. Upon ingestion by humans—often through the consumption of raw or undercooked seafood—V. parahaemolyticus senses the host environment and expresses numerous genes, the products of which synergize to synthesize and secrete toxins that can cause acute gastroenteritis. To understand the regulation of such adaptive response, mRNA transcripts must be mapped accurately. However, due to the limitations of common sequencing methods, not all features of bacterial transcriptomes are always reported. We applied direct RNA sequencing to analyze the V. parahaemolyticus transcriptome. Mapping the full features of the transcriptome is anticipated to enhance our understanding of gene regulation in this bacterium and provides a data set for future work. Additionally, this study reveals a deeper view of a complicated transcriptome landscape, demonstrating the importance of applying such methods to other bacterial models.

Despite many advantages over injection vaccines such as cost effectiveness, safety and easy to use, and so on, oral vaccines are negligibly concerned. This is mostly because of the availability of vast surface in the gastro-intestinal tract, thereby requiring lot of antigens which could hamper their potential. To circumvent this issue, a novel strategy for targeting antigens to M cells (microfold cells), a minority of cells located in the small intestine for antigen transportation, is utilized by making a fusion protein comprised of an antigen with an M cell specific ligand. Discovered via biopanning, Co1 peptide is a potential ligand because of its small size (12 amino acids) and having an adjuvant capacity. To develop a monitoring model, we fused GFP (green fluorescent protein) as a monitoring marker with Co1 peptide. Initially, co1-gfp fusion gene was created by overlap extension PCR on GFP-encoded vector backbone, then it was incorporated into an expression vector pET22b before transforming into E. coli DH5α. The recombinant vector was screened by PCR method, sequenced and aligned with designed sequences. The in-frame vector was then introduced into E. coli BL21(DE3) for expression by inducing with 0.5mM IPTG. Fusion protein was purified using Ni-affinity chromatography. The results showed that the fused genes were in-frame cloned and completely matched with the designed sequences. SDS-PAGE and Western blot analyses showed Co1-GFP protein expressed in soluble form and could be purified at one-step elution of 500mM imidazole. The purified fusion protein could emit fluorescent light under UV excitation. Collectively, recombinant Co1-GFP fusion protein was successfully produced and its potential applications need to be warranted.

With a production of 39 million metric tons each year, rice is one of the main agricultural products of Vietnam. Thus, rice straw is a significant by-product, whose use in a biorefinery process would contribute to the bio-based transformation of the Vietnamese and South East Asian economy. In order to find novel efficient enzyme mixtures for the hydrolysis of rice straw and other agricultural residues, we took advantage of the rapidly evolving biodiversity of Vietnam and screened 1100 new fungal isolates from soil and decaying plant tissues for their CMCase activity. We selected 36 strains and evaluated them for their cellulases, xylanases, and accessory enzymes activities. Most of these isolates belonged to the genera Aspergillus and Trichoderma. We identified a few promising isolates, such as A. brunneoviolaceus FEC 156, A. niger FEC 130 and FEC 705, and A. tubingensis FEC 98, FEC 110 and FEC 644, whose produced enzyme mixtures released a mass fraction of the sugar content of alkali-treated rice straw higher than 20%, compared to 10% for Trichoderma reesei RUT C-30. We verified that the black Aspergilli are particularly efficient in their saccharification ability. We also identified strains that although they produced low amounts of cellulases and xylanases, their enzyme mixtures had high saccharification efficiencies, indicating the importance of the synergy effect, rather than the amount of enzymes available. Our results highlight the intra-species variation, especially in the Trichoderma genus, regarding the biomass degradation characteristics and the associated range of enzymatic activities. (C) 2017 The Author(s). Published by Elsevier Ltd.

Fish raised in aquaculture ponds may get infected with fishborne zoonotic trematodes (FZT) during the nursing stage. Freshwater snails serve as intermediate hosts for FZT and we wanted to explore the possibility of controlling snails by stocking nursery ponds with a few juvenile specimens of the mollusc-eating fish, black carp ( Mylopharyngodon piceus ). Obviously, the risk that black carp might also prey on the juvenile fishes in nursery ponds should first be assessed. Laboratory trials showed that all size classes of juvenile black carp consumed fry of common carp ( Cyprinus carpio ) even when offered snails as food; the odds of survival of fry from tanks with medium sized and large black carp was 5.6% and 39.9%, respectively of that of fry in tanks with small sized black carp. Since the large black carp also consumed fewer snails than medium sized fish, we believe that large specimens were stressed in the experimental aquaria. Under semi-field conditions, presence of the black carp had no effect on survival of fry of Oreochromis niloticus and C. carpio both in the absence and presence of snails as alternative food. The black carp consumed most snails offered with the exception of some of the large snails. We conclude that under field conditions, predation by black carp on fish fry is minimal and field trials in nursery ponds are warranted. Due to the risks that black carp pose to native imperiled snails and other molluscs, trials should be restricted to ponds within the fish’s native or existing range.

Freshwater snails of the family Lymnaeidae play an important role in the transmission of fascioliasis worldwide. In Vietnam, 2 common lymnaeid species, Lymnaea swinhoei and Lymnaea viridis, can be recognized on the basis of morphology, and a third species, Lymnaea sp., is known to exist. Recent studies have raised controversy about their role in transmission of Fasciola spp. because of confusion in identification of the snail hosts. The aim of this study is, therefore, to clarify the identities of lymnaeid snails in Vietnam by a combination of morphological and molecular approaches. The molecular analyses using the second internal transcribed spacer (ITS2) of the nuclear ribosomal DNA clearly showed that lymnaeids in Vietnam include 3 species, Austropeplea viridis (morphologically identified as L. viridis), Radix auricularia (morphologically identified as L. swinhoei) and Radix rubiginosa (morphologically identified as Lymnaea sp.). R. rubiginosa is a new record for Vietnam. Among them, only A. viridis was found to be infected with Fasciola spp. These results provide a new insight into lymnaeid snails in Vietnam. Identification of lymnaeid snails in Vietnam and their role in the liver fluke transmission should be further investigated.

Fish-borne zoonotic trematodes, such as Clonorchis sinensis, heterophyids and others, constitute a public health concern in parts of northern Vietnam and infections with these trematodes are often thought to be linked to fish culture. One common fish culture system is the integrated fish-livestock (VAC) ponds where individual households have 1 or more ponds. Fish fry, mainly of various carp species, produced in hatcheries, not necessarily local, are introduced into nursery ponds and after approximately 6 weeks, juvenile fishes are transferred to household ponds, referred to as grow-out ponds. Grow-out ponds are usually fertilized with organic debris, including animal excreta, to stimulate algal growth and subsequently fish growth. This paper describes the distribution of freshwater snails and occurrence of trematode infections in these in VAC ponds and associated habitats as part of a major study on risk factors of FZT infections in cultured fish in two communes, Nghia Lac and Nghia Phu, Nghia Hung District, Nam Dinh Province. The area is under intense rice cultivation with an extensive canal network supplying fields and also household VAC ponds. A total of 16 snail species was found and four were widely distributed i.e. Angulyagra polyzonata, Melanoides tuberculata, Bithynia fuchsiana and Pomacea insularum. Snail diversity and counts were higher in nursery ponds than in grow-out ponds. Species of the families Thiaridae and Viviparidae were more abundant than other species in VAC ponds while species of the Bithyniidae, Stenothyridae and Planorbidae dominated in rice fields and small canals. Trematode infections were found in eight snail species and among these M. tuberculata had the highest overall prevalence of infection (13.28%). No trematode infections were found in species of the Viviparidae and Ampullaridae except for metacercariae. Parapleurolophocercous and pleurolophocercous cercariae constituted the most common type of cercariae recovered, contributing 40.6% of all infections followed by echinostome cercariae (35.0%) and xiphidiocercariae (17.3%). Bithynia fuschiana and M. tuberculata had the most diverse trematode fauna. C. sinensis was not recorded in this study. The VAC pond system in this area, is very important for transmission of minute intestinal trematodes while they play little role in transmission of C. sinensis as its intermediate hosts, bithynid snails, rarely occur in these ponds. From a public health perspective this is positive as the effects of infections with intestinal trematodes are considered mild. On the other hand it is possible that even such subtle effects could have importance in public health as transmission is very intense in the area. And this in combination with the aquaculture importance, reduced marketability of fishes with high metacercariae loads, warrants that control efforts against these trematodes are initiated to reduce transmission in this production system.

Paragonimosis is an important food-borne zoonosis especially in Asian countries. Among Paragonimus species, Paragonimus westermani followed by P. skrjabini complex are the major pathogens for human paragonimosis in Asia. In addition, P. heterotremus is an important pathogen in southern China and the Indochina Peninsula and is the only proven species to cause human paragonimosis in Vietnam. During a recent survey in Yenbai Province in northern Vietnam, we found small and large types of Paragonimus metacercariae often concurrently in mountainous crabs, Potamiscus tannanti. Adult worms from those small and large metacercariae were obtained separately by experimental infection in dogs and cats. Morphological and molecular phylogenetic study based on sequences of ITS2 and a part of CO1 genes were performed for the identification of small and large metacercariae and their adults. The results showed that small metacercariae and their adults are completely identical with P. heterotremus in morphology and molecular genetic profiles. In contrast, large metacercariae and their adults have some morphological similarities with P. skrjabini and P. harinasutai, but are unidentifiable from each other by morphology alone. Molecular phylogenetic tree analyses on ITS2 and CO1 genes revealed that large metacercariae and their adults were grouped in the same clade and different from any known Paragonimus species. Although they share the same ancestor with P. skrjabini complex, their genetic distance was considerably different from two other known subspecies, P. skrjabini skrjabini and P. skrjabini miyazakii. Our results provide a new insight on the phylogeny of the genus Paragonimus.