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Parasitology Research Dec 2013Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where...
Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4 years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.
Topics: Animals; DNA; Genome, Helminth; Genotyping Techniques; Humans; Larva; Microsatellite Repeats; Polymerase Chain Reaction; Schistosoma japonicum; Sequence Analysis, DNA; Specimen Handling
PubMed: 24013341
DOI: 10.1007/s00436-013-3587-9 -
Parasitology Research Jul 2015More than 40 kinds of mammals in China are known to be naturally infected with Schistosoma japonicum (S. japonicum) (Peng et al. Parasitol Res 106:967-76, 2010).... (Comparative Study)
Comparative Study
More than 40 kinds of mammals in China are known to be naturally infected with Schistosoma japonicum (S. japonicum) (Peng et al. Parasitol Res 106:967-76, 2010). Compared with permissive BALB/c mice, rats are less susceptible to S. japonicum infection and are considered to provide an unsuitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), via the regulation of gene expression at the transcriptional and post-transcriptional levels, may be responsible for developmental differences between schistosomula in these two rodent hosts. Solexa deep-sequencing technology was used to identify differentially expressed miRNAs from schistosomula isolated from Wistar rats and BALB/c mice 10 days post-infection. The deep-sequencing analysis revealed that nearly 40 % of raw reads (10.37 and 10.84 million reads in schistosomula isolated from Wistar rats and BALB/c mice, respectively) can be mapped to selected mirs in miRBase or in species-specific genomes. Further analysis revealed that several miRNAs were differentially expressed in schistosomula isolated from these two rodents; 18 were downregulated (by <2-fold) and 23 were up-regulated (>2-fold) (expression levels in rats compare with those in mice). Additionally, three novel miRNAs were primarily predicted and identified. Among the 41 differentially expressed miRNAs, 4 miRNAs had been identified with specific functions in schistosome development or host-parasite interaction, such as sexual maturation (sja-miR-1, sja-miR-7-5p), embryo development (sja-miR-36-3p) in schistosome, and pathogenesis of schistosomiasis (sja-bantam). Then, the target genes were mapped, filtered, and correlated with a set of genes that were differentially expressed genes in schistosomula isolated from mice and rats, which we identified in a S. japonicum oligonucleotide microarray analysis in a previous study. Gene Ontology (GO) analysis of the predicted target genes of 13 differentially expressed miRNAs revealed that they were involved in some important biological pathways, such as metabolic processes, the regulation of protein catabolic processes, catalytic activity, oxidoreductase activity, and hydrolase activity. The study presented here includes the first identification of differentially expressed miRNAs between schistosomula in mice or rats. Therefore, we hypothesized that the differentially expressed miRNAs may affect the development, growth, and maturation of the schistosome in its life cycle. Our analysis suggested that some differentially expressed miRNAs may impact the survival and development of the parasite within a host. This study increases our understanding of schistosome development and host-parasite interactions.
Topics: Animals; Computational Biology; Female; Gene Expression Regulation; Gene Library; Host-Parasite Interactions; Life Cycle Stages; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Rats; Rats, Wistar; Schistosoma japonicum; Schistosomiasis japonica; Sequence Analysis, DNA
PubMed: 25895062
DOI: 10.1007/s00436-015-4468-1 -
Parasites & Vectors Sep 2011Tetraspanins (TSPs), also known as members of the trans-membrane 4 super-family (TM4SF), comprise an assemblage of surface antigens reported in eukaryotic organisms. In...
BACKGROUND
Tetraspanins (TSPs), also known as members of the trans-membrane 4 super-family (TM4SF), comprise an assemblage of surface antigens reported in eukaryotic organisms. In the work presented here, six novel TSP proteins from the human blood fluke Schistosoma japonicum (S. japonicum) were produced and analyzed through a combination of bioinformatics and experimental approaches.
RESULTS
Six novel TSP proteins of Schistosoma japonicum (designated as Sj-TSP-#1~6) contained four trans-membrane regions and one large extracellular loop (LEL) with a conserved CCG motif. Size of the proteins varied from 227 to 291 amino acid residues. All the six proteins were produced in E.coli and immune sera to each protein were prepared. Analysis of transcription profiles of the proteins by RT-PCR showed that Sj-TSP-#4 was transcribed only in the egg stage while transcription of the Sj-TSP-#2 was detected in female worms but not in males. The similar results were obtained by Western blot. Immunolocalization of the TSP proteins by immunofluorescence assay showed that the Sj-TSP-#2, Sj-TSP-#5 and Sj-TSP-#6 were located in the tegument of worms.
CONCLUSIONS
This study provided six novel TSP members of S. japonicum including their sequences and recombinant proteins. Availability of the novel proteins and information on their expression profile and location provided a basis for further investigation of the TSP proteins for their biological functions and as vaccine candidates.
Topics: Amino Acid Sequence; Animals; Female; Gene Expression Regulation, Developmental; Helminth Proteins; Humans; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Phylogeny; Protein Structure, Tertiary; Protein Transport; Schistosoma japonicum; Schistosomiasis japonica; Sequence Alignment; Species Specificity; Tetraspanins
PubMed: 21958506
DOI: 10.1186/1756-3305-4-190 -
PLoS Computational Biology Oct 2014Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other host-parasite interactions. Hundreds of genes...
Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other host-parasite interactions. Hundreds of genes encoding putative proteases have been identified in the recently published schistosome genomes. However, the expression profiles of these proteases in Schistosoma species have not yet been systematically analyzed. We retrieved and culled the redundant protease sequences of Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, and Clonorchis sinensis from public databases utilizing bioinformatic approaches. The degradomes of the four parasitic organisms and Homo sapiens were then comparatively analyzed. A total of 262 S. japonicum protease sequences were obtained and the expression profiles generated using whole-genome microarray. Four main clusters of protease genes with different expression patterns were identified: proteases up-regulated in hepatic schistosomula and adult worms, egg-specific or predominantly expressed proteases, cercaria-specific or predominantly expressed proteases, and constantly expressed proteases. A subset of protease genes with different expression patterns were further validated using real-time quantitative PCR. The present study represents the most comprehensive analysis of a degradome in Schistosoma species to date. These results provide a firm foundation for future research on the specific function(s) of individual proteases and may help to refine anti-proteolytic strategies in blood flukes.
Topics: Animals; Cathepsins; Cluster Analysis; Female; Gene Expression Profiling; Helminth Proteins; Humans; Male; Oligonucleotide Array Sequence Analysis; Peptide Hydrolases; Phylogeny; Platyhelminths; Schistosoma japonicum
PubMed: 25275570
DOI: 10.1371/journal.pcbi.1003856 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Aug 2021To develop a rapid test for detection of specific gene fragments based on the recombinase-aided isothermal amplification assay (RAA) and nucleic acid dipstick test.
OBJECTIVE
To develop a rapid test for detection of specific gene fragments based on the recombinase-aided isothermal amplification assay (RAA) and nucleic acid dipstick test.
METHODS
The gene fragment was selected as the target gene fragment, and the primers and fluorescent probe were designed and synthesized. Then, a nucleic acid dipstick test was established. The sensitivity of this dipstick test was evaluated by detecting different copies of recombinant plasmids containing the gene fragment and different concentrations of genomic DNA from adult worms of , and the specificity of the dipstick test was evaluated by detecting the genomic DNA from , , , , and .
RESULTS
The nucleic acid dipstick test based on the gene fragment showed the minimum detectable limit of 10 copies/μL of the recombinant plasmid containing the gene fragment and the minimum detectable limit of 1 pg/μL of genomic DNA, and the dipstick assay tested negative for the genomic DNA from , , , , and .
CONCLUSIONS
A rapid, simple, and visualized assay is established for detection of specific gene fragments based on RAA and nucleic acid dipstick test.
Topics: Animals; Nucleic Acid Amplification Techniques; Nucleic Acids; Recombinases; Schistosoma japonicum; Sensitivity and Specificity
PubMed: 34505438
DOI: 10.16250/j.32.1374.2021016 -
Parasitology Jan 1997This paper describes the localization of paramyosin immunoreactivity in Schistosoma japonicum and represents the first comparative immunolocalization study among...
This paper describes the localization of paramyosin immunoreactivity in Schistosoma japonicum and represents the first comparative immunolocalization study among schistosome adult, cercariae and lung schistosomula by electron microscopy. A polyclonal antibody was utilized to immunolabel paramyosin or paramyosin-like proteins. Paramyosin was localized within the muscle layer of all 3 developmental stages. Furthermore, paramyosin was localized within granules of the post-acetabular glands of cercariae, and within the tegument matrix and surface of lung schistosomules. Adults and cercariae did not display any detectable paramyosin on the surface or within the tegument. The possible functions of paramyosin within S. japonicum and the relevance of these findings in relation to the reported protective properties of paramyosin as an anti-schistosome vaccine target molecule are discussed.
Topics: Animals; Female; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Microscopy, Immunoelectron; Schistosoma japonicum; Schistosomiasis japonica; Snails; Tropomyosin
PubMed: 9011073
DOI: 10.1017/s0031182096008001 -
PLoS Neglected Tropical Diseases 2015Schistosoma japonicum causes major public health problems in China and the Philippines; this parasite, which is transmitted by freshwater snails of the species... (Comparative Study)
Comparative Study
BACKGROUND
Schistosoma japonicum causes major public health problems in China and the Philippines; this parasite, which is transmitted by freshwater snails of the species Oncomelania hupensis, causes the disease intestinal schistosomiasis in humans and cattle. Researchers working on Schistosoma in Africa have described the relationship between the parasites and their snail intermediate hosts as coevolved or even as an evolutionary arms race. In the present study this hypothesis of coevolution is evaluated for S. japonicum and O. hupensis. The origins and radiation of the snails and the parasite across China, and the taxonomic validity of the sub-species of O. hupensis, are also assessed.
METHODOLOGY/PRINCIPAL FINDINGS
The findings provide no evidence for coevolution between S. japonicum and O. hupensis, and the phylogeographical analysis suggests a heterochronous radiation of the parasites and snails in response to different palaeogeographical and climatic triggers. The results are consistent with a hypothesis of East to West colonisation of China by Oncomelania with a re-invasion of Japan by O. hupensis from China. The Taiwan population of S. japonicum appears to be recently established in comparison with mainland Chinese populations.
CONCLUSIONS/SIGNIFICANCE
The snail and parasite populations of the western mountain region of China (Yunnan and Sichuan) appear to have been isolated from Southeast Asian populations since the Pleistocene; this has implications for road and rail links being constructed in the region, which will breach biogeographical barriers between China and Southeast Asia. The results also have implications for the spread of S. japonicum. In the absence of coevolution, the parasite may more readily colonise new snail populations to which it is not locally adapted, or even new intermediate host species; this can facilitate its dispersal into new areas. Additional work is required to assess further the risk of spread of S. japonicum.
Topics: Animals; Biological Evolution; China; Humans; Japan; Molecular Sequence Data; Phylogeny; Phylogeography; Schistosoma japonicum; Schistosomiasis; Snails
PubMed: 26230619
DOI: 10.1371/journal.pntd.0003935 -
Parasites & Vectors Oct 2017Schistosomiasis is one of the most common parasitic diseases affecting millions of humans and animals worldwide. Understanding the signal transduction pathways and the...
BACKGROUND
Schistosomiasis is one of the most common parasitic diseases affecting millions of humans and animals worldwide. Understanding the signal transduction pathways and the molecular basis of reproductive regulation in schistosomes is critically important for developing new strategies for preventing and treating these infections. Syk kinases regulate the proliferation, differentiation, morphogenesis, and survival of various types of cells and have been identified in invertebrates. Tyrosine kinase 4 (TK4), a member of the Syk kinase family, plays a pivotal role in gametogenesis in S. mansoni, affecting the development of the testis and ovaries in this parasite. The role of TK4, however, in the reproduction of S. japonicum is poorly understood.
METHODS
Here, the complete coding sequence of TK4 gene in S. japonicum (SjTK4) was cloned and characterized. The expression of SjTK4 was analyzed at different life-cycle stages and in various tissues of S. japonicum by qPCR. Piceatannol, a Syk kinase inhibitor, was applied to S. japonicum in vitro. The piceatannol-induced morphological changes of the parasites were observed using confocal laser scanning microscopy and the alterations in important egg-shell synthesis-related genes were examined using qPCR analyses.
RESULTS
SjTK4 mRNA was differentially expressed throughout the life-cycle of S. japonicum. SjTK4 mRNA was highly expressed in the ovary and testis of S. japonicum, with the level of gene expression significantly higher in males than in females. The expression levels of some important egg-shell synthesis related genes were higher in the piceatannol-treated groups than in the vehicle-treated control group and the number of eggs and germ cells also decreased in a concentration-dependent manner. Importantly, large pore-like structures can be found in the testis and ovaries of males and females after treating with piceatannol.
CONCLUSION
The results suggest that SjTK4 may play an important role in regulating gametogenesis of S. japonicum. The findings may help better understand the fundamental biology of S. japonicum. Moreover, the effect of S. japonicum treatment by piceatannol provides us with a new idea that inhibition of SjTK4 signaling pathway can effectively retard the development of the testis and ovaries.
Topics: Animals; Coinfection; Female; Humans; Male; Mice; Protein-Tyrosine Kinases; Reproduction; Schistosoma japonicum; Schistosomiasis japonica; Stilbenes
PubMed: 29047397
DOI: 10.1186/s13071-017-2453-5 -
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng... 1989
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Mitochondrial DNA Feb 2015The present study examined sequence variability in the mitochondrial (mt) protein-coding genes cytochrome b (cytb), NADH dehydrogenase subunits 2 and 6 (nad2 and nad6)...
The present study examined sequence variability in the mitochondrial (mt) protein-coding genes cytochrome b (cytb), NADH dehydrogenase subunits 2 and 6 (nad2 and nad6) among 24 isolates of Schistosoma japonicum from different endemic regions in the Philippines, Japan and China. The complete cytb, nad2 and nad6 genes were amplified and sequenced separately from individual schistosome. Sequence variations for isolates from the Philippines were 0-0.5% for cytb, 0-0.6% for nad2, and 0-0.9% for nad6. Variation was 0-0.5%, 0.1-0.8%, 0-0.7% for corresponding genes for schistosome samples from mainland China. For worms in Japan, genetic variations were 0-0.2%, 0.1-0.2% and 0 for the three genes, respectively. Sequence variations were 0-1.0%, 0-1.8% and 0-1.1% for cytb, nad2 and nad6, respectively, among schistosome isolates from different geographical strains in the Philippines, Japan and China. Of the three countries, lowest sequence variations were found between isolates from mainland China and the Philippines and highest were detected between Japan and the Philippines in three mtDNA genes. Phylogenetic analyses based on the combined sequences of cytb, nad2 and nad6 revealed that all isolates in the Philippines clustered together sistered to samples from Yunnan and Zhejiang provinces in China, while isolates from Yamanashi in Japan were in a solitary clade. These results demonstrated the usefulness of the combined three mtDNA sequences for studying genetic diversity and population structure among S. japonicum isolates from the Philippines, China and Japan.
Topics: Animals; China; Female; Genes, Mitochondrial; Genetic Variation; Japan; Male; Philippines; Phylogeny; Phylogeography; Schistosoma japonicum; Sequence Analysis, DNA
PubMed: 23901927
DOI: 10.3109/19401736.2013.814110