[Establishment of Duplex PCR for Identifying Metagonimus yokogawai and Haplorchis taichui].

OBJECTIVE: To develop a duplex PCR method for identifying Metagonimus yokogawai and Haplorchis taichui. METHODS: ITS1 sequences of M. yokogawai and H. taichui, as well as those of their homologous species were obtained from GenBank, and two sets of specific primer pairs for M. yokogawai and H. taichui were designed accordingly using Primer Premier 5.0 software. PCR reaction system and conditions were optimized. The established duplex PCR method was applied in a pool of M. yokogawai, H. taichui, and 17 related species to examine its specificity. Sensitivity was evaluated through serial dilutions of plasmids containing their specific sequences. Finally, the duplex PCR was applied to identify M. yokogawai and H. taichui among trematodes collected from the viscera of 47 cats and 40 dogs to test its practicality. RESULTS: The duplex PCR method amplified target sequences of M. yokogawai and H. taichui, generating 648 bp and 279 bp products, respectively. No cross reaction was found with the following 17 related species: Haplorchis pumilio, Clonorchis sinensis, Pharyngostomum cordatum, the metacercaria of Metorchis sp. and Exorchis sp., Echinochasmus liliputanus, Echinochasmus perfoliatus, Echinostoma friedi, Hypoderaeum conoideum, Holostephanus sp., Diplodiscus sp., Anisakis sp., Metorchis orientais, Paragonimus westermani, Watsonius watsoni, Notocotylus sp. and Hysterothylacium sp, indicating a high specificity of this method. The detection limits for DNAs of M. yokogawai and H. taichui were 1.49 x 10(-1) pg and 1.14 x 10(-1) pg, suggesting a good sensitivity for this method. Further, the duplex PCR successfully identified M. yokogawai and H. taichui from cat and dog viscera, with no cross amplification of other trematodes. CONCLUSION: The duplex PCR is effective in identifying Metagonimus yokogawai and Haplorchis taichui.

Dysregulation of hepatic microRNA expression in C57BL/6 mice affected by excretory-secretory products of Fasciola gigantica.

The excretory-secretory products released by the liver fluke Fasciola gigantica (FgESPs) play important roles in regulating the host immune response during the infection. Identification of hepatic miRNAs altered by FgESPs may improve our understanding of the pathogenesis of F. gigantica infection. In this study, we investigated the alterations in the hepatic microRNAs (miRNAs) in mice treated with FgESPs using high-throughput small RNA (sRNA) sequencing and bioinformatics analysis. The expression of seven miRNAs was confirmed by quantitative stem-loop reverse transcription quantitative PCR (qRT-PCR). A total of 1,313 miRNAs were identified in the liver of mice, and the differentially expressed (DE) miRNAs varied across the time lapsed post exposure to FgESPs. We identified 67, 154 and 53 dysregulated miRNAs at 1, 4 and 12 weeks post-exposure, respectively. 5 miRNAs (miR-126a-3p, miR-150-5p, miR-155-5p, miR-181a-5p and miR-362-3p) were commonly dysregulated at the three time points. We also found that most of the DE miRNAs were induced by FgESPs in the mouse liver after 4 weeks of exposure. These were subjected to Gene Ontology (GO) enrichment analysis, which showed that the predicted targets of the hepatic DE miRNAs of mice 4 weeks of FgESPs injection were enriched in GO terms, including cell membrane, ion binding, cellular communication, organelle and DNA damage. KEGG analysis indicated that the predicted targets of the most downregulated miRNAs were involved in 15 neural activity-related pathways, 6 digestion-related pathways, 20 immune response-related pathways and 17 cancer-related pathways. These data provide new insights into how FgESPs can dysregulate hepatic miRNAs, which play important roles in modulating several aspects of F. gigantica pathogenesis.

DNA methylation and hydroxymethylation profiles reveal possible role of highly methylated TLR signaling on Fasciola gigantica excretory/secretory products (FgESPs) modulation of buffalo dendritic cells.

BACKGROUND: Excretory/secretory products (ESPs) released by parasites influence the development and functions of host dendritic cells (DCs). However, little is known about changes of DNA (hydroxy)methylation on DC development during Fasciola gigantica infection. The present study aimed to investigate whether F. gigantica ESPs (FgESPs) affects the development and functions of buffalo DCs through altering the DNA (hydroxy)methylation of DCs. METHODS: Buffalo DCs were prepared from peripheral blood mononuclear cells (PBMCs) and characterized using scanning and transmission electron microscopy (SEM/TEM) and quantitative reverse transcriptional PCR (qRT-RCR). DCs were treated with 200 µg/ml of FgESPs in vitro, following DNA extraction. The DNA methylome and hydroxymethylome were profiled based on (hydroxy)methylated DNA immunoprecipitation sequencing [(h)MeDIP-Seq] and bioinformatics analyses. qRT-RCR was also performed to assess the gene transcription levels of interest. RESULTS: FgESPs markedly suppressed DC maturation evidenced by morphological changes and downregulated gene expression of CD1a and MHC II. Totals of 5432 and 360 genes with significant changes in the 5-methylcytosine (5-mC) and the 5-hydroxymethylcytosine (5-hmC) levels, respectively, were identified in buffalo DCs in response to FgESPs challenge. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these differentially expressed genes were highly enriched in pathways associated with immune response. Some cancer-related pathways were also indicated. There were 111 genes demonstrating changes in both 5-mC and 5-hmC levels, 12 of which were interconnected and enriched in 12 pathways. The transcription of hypermethylated genes TLR2, TLR4 and IL-12B were downregulated or in a decreasing trend, while the mRNA level of high-hydroxymethylated TNF gene was upregulated in buffalo DCs post-exposure to FgESPs in vitro. CONCLUSIONS: To our knowledge, the present study provides for the first time a unique genome-wide profile of DNA (hydroxy)methylation for DCs that interact with FgESPs, and suggests a possible mechanism of FgESPs in suppressing DC maturation and functions that are involved in TLR signaling.