[show abstract][hide abstract] ABSTRACT: Heme is an essential molecule for vast majority of organisms serving as a prosthetic group for various hemoproteins. Although most organisms synthesize heme from 5-aminolevulinic acid through a conserved heme biosynthetic pathway composed of seven consecutive enzymatic reactions, nematodes are known to be natural heme auxotrophs. The completely sequenced Caenorhabditis elegans genome, for example, lacks all seven genes for heme biosynthesis. However, genome/transcriptome sequencing of Strongyloides venezuelensis, an important model nematode species for studying human strongyloidiasis, indicated the presence of a gene for ferrochelatase (FeCH), which catalyzes the terminal step of heme biosynthesis, whereas the other six heme biosynthesis genes are apparently missing. Phylogenetic analyses indicated that nematode FeCH genes, including that of S. venezuelensis (SvFeCH) have a fundamentally different evolutionally origin from the FeCH genes of non-nematode metazoa. Although all non-nematode metazoan FeCH genes appear to be inherited vertically from an ancestral opisthokont, nematode FeCH may have been acquired from an alpha-proteobacterium, horizontally. The identified SvFeCH sequence was found to function as FeCH as expected based on both in vitro chelatase assays using recombinant SvFeCH and in vivo complementation experiments using an FeCH-deficient strain of Escherichia coli. Messenger RNA expression levels during the S. venezuelensis lifecycle were examined by real-time RT-PCR. SvFeCH mRNA was expressed at all the stages examined with a marked reduction at the infective third-stage larvae. Our study demonstrates the presence of a bacteria-like FeCH gene in the S. venezuelensis genome. It appeared that S. venezuelensis and some other animal parasitic nematodes reacquired the once-lost FeCH gene. Although the underlying evolutionary pressures that necessitated this reacquisition remain to be investigated, it is interesting that the presence of FeCH genes in the absence of other heme biosynthesis genes has been reported only for animal pathogens, and this finding may be related to nutritional availability in animal hosts.
PLoS ONE 01/2013; 8(3):e58458. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Strongyloides venezuelensis is one of some 50 species of genus Strongyloides, obligate gastrointestinal parasites of vertebrates, responsible for strongyloidiasis in humans and other domestic/companion animals. Although S. venezuelensis has been widely used as a model species for studying human/animal strongyloidiasis, the sequence information for this species has been quite limited. To create a more comprehensive catalogue of expressed genes for identification of genes potentially involved in animal parasitism, we conducted a de novo sequencing analysis of the transcriptomes from four developmental stages of S. venezuelensis, using a Roche 454 GS FLX Titanium pyrosequencing platform. A total of 14,573 contigs were produced after de novo assemblies of over 2million sequencing reads and formed a dataset "Vene454". BLAST homology search of Vene454 against proteome and transcriptome data from other animal-parasitic and non-animal-parasitic nematode species revealed several interesting genes, which may be potentially related to animal parasitism, including nicotinamide phosphoribosyltransferase and ferrochelatase. The Vene454 dataset analysis also enabled us to identify transcripts that are specifically enriched in each developmental stage. This work represents the first large-scale transcriptome analysis of S. venezuelensis and the first study to examine the transcriptome of the lung L3 developmental stage of any Strongyloides species. The results not only will serve as valuable resources for future functional genomics analyses to understand the molecular aspects of animal parasitism, but also will provide essential information for ongoing whole genome sequencing efforts in this species.
Parasitology International 09/2012; · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vibrio nigripulchritudo is considered one of the major pathogens threatening shrimp aquaculture. In this study, we developed a novel and highly specific
quantitative loop-mediated isothermal amplification (Q-LAMP) assay. A set of four specific primers were designed targeting
the V. nigripulchritudo intergenic spacer region. The reaction time and temperature were optimized for 60min at 63°C. Quantitative analysis was
then performed by measuring the turbidity of the reaction solution using a real-time turbidimeter, allowing for quantification
of the initial DNA concentration with a sensitivity of 102 copy numbers equivalent to 2.3 colony forming units/ml or 0.3fg/μl. The LAMP assay was able to specifically detect two representative
strains of V. nigripulchritudo, whereas other Vibrio and non-Vibrio species were not amplified. A standard curve was generated for V. nigripulchritudo by plotting the threshold time (T
t) versus the log of bacterial number. A high correlation coefficient (R
2=0.9749) was observed for the Q-LAMP reaction. In conclusion, Q-LAMP assay is a sensitive, rapid, and simple tool that can
be used for the detection and quantification of V. nigripulchritudo in shrimp, thereby facilitating surveillance of vibriosis infection.
KeywordsQuantitative loop-mediated isothermal amplification–
–Intergenic spacer region–DNA
[show abstract][hide abstract] ABSTRACT: Human interferon-alpha (huIFN-alpha) is an important immunomodulatory substance used in the treatment and prevention of numerous infectious and immune-related diseases in animals. However, the immunostimulatory effects of huIFN-alpha in fish remain to be investigated. In the current study, the immune responses of the carp species Cyprinus carpio L. to treatment with huIFN-alpha were analyzed via measurement of superoxide anion production, phagocytic activity and the expression of cytokine genes including interleukin-1beta, tumor necrosis factor-alpha and interleukin 10. Low doses of huIFN-alpha were administered orally once a day for 3 days, and sampling was carried out at 1, 3 and 5 days post-treatment. Our results indicate that a low dose of huIFN-alpha significantly increased phagocytic activity and superoxide anion production in the carp kidney. The huIFN-alpha-treated fish also displayed a significant upregulation in cytokine gene expression. The current study demonstrates the stimulatory effects of huIFN-alpha on the carp immune system and highlights the immunomodulatory role of huIFN-alpha in fish.
Veterinary Immunology and Immunopathology 05/2009; 131(3-4):273-7. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: LAMP is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a diagnostic protocol was developed for the detection of Vibrio nigripulchritudo in shrimps. Vibrio nigripulchritudo is associated with distinct shrimp diseases (vibriosis) and is considered one of the threatening pathogens in shrimp industry. After initial cloning and sequencing of the intergenic spacer region (ITS) between 16S and 23S rRNA genes of V. nigripulchritudo, a set of four primers - two inner and two outer - were designed for use in the LAMP reaction. Reaction time and temperature were optimized for 60 min at 63 degrees C, respectively. The detection limit of V. nigripulchritudo by LAMP was 10(2) CFU mL(-1) but PCR could detect up to 10(3) CFU mL(-1). The LAMP method could detect the presence of V. nigripulchritudo from heart, lymphoid organ, and muscle of experimentally infected shrimps with V. nigripulchritudo. This study established a highly sensitive and a rapid diagnostic procedure for detection of V. nigripulchritudo in shrimps. The method developed in this study could be very useful for routine shrimp disease diagnostics.