During the past several decades, researches on parasite genetics have progressed from biochemical and serodiagnostic studies to protein chemistry, molecular biology, and functional gene studies. Nowadays, bioinformatics, genomics, and proteomics approaches are being applied by Korean parasitology researchers. As for Clonorchis sinensis, investigations have been carried out to identify its functional genes using forward and reverse genetic approaches and to characterize the biochemical and biological properties of its gene products. The authors review the proteins of cloned genes, which include antigenic proteins, physiologic and metabolic enzymes, and the gene expression profile of Clonorchis sinensis.
Animals ; Clonorchiasis/parasitology ; Clonorchis sinensis/enzymology/*genetics/*metabolism ; Gene Expression Regulation ; Helminth Proteins/*genetics/*metabolism ; Humans

OBJECTIVE: To evaluate the performance of recombinase-aided amplification (RAA) assay in detection of Clonorchis sinensis metacercariae in freshwater fish samples, so as to provide insights into standardization and field application of this assay. METHODS: Wild freshwater fish samples were collected in the rivers of administrative villages where C. sinensis-infected residents lived in Jiangyan District, Xinghua County and Taixing County of Taizhou City, Jiangsu Province from June to September 2022. Genomic DNA was extracted from six freshwater fish specimens (5 g each) containing 0, 1, 2, 4, 8 and 16 C. sinensis metacercariae for fluorescent RAA assay, and the diagnostic sensitivity was evaluated. Fluorescent RAA assay was performed with genomic DNA from C. sinensis, Metorchis orientalis, Haplorchis pumilio and Centrocestus formosanus metacercariae as templates to evaluate its cross-reactions. In addition, the detection of fluorescent RAA assay and direct compression method for C. sinensis metacercariae was compared in field-collected freshwater fish samples. RESULTS: Positive amplification was found in fresh-water fish specimens containing different numbers of C. sinensis metacercariae, and fluorescent RAA assay was effective to detect one C. sinensis metacercaria in 5 g freshwater fish specimens within 20 min. Fluorescent RAA assay tested negative for DNA from M. orientalis, H. pumilio and C. formosanus metacercariae. Fluorescent RAA assay and direct compression method showed 5.36% (93/1 735) and 2.88% (50/1 735) detection rates for C. sinensis metacercariae in 1 735 field-collected freshwater fish samples, with a statistically significant difference seen (χ² = 478.150, P < 0.001). There was a significant difference in the detection of C. sinensis metacercariae in different species of freshwater fish by both the direct compression method (χ² = 11.20, P < 0.05) and fluorescent RAA assay (χ² = 20.26, P < 0.001), and the detection of C. sinensis metacercariae was higher in Pseudorasbora parva than in other fish species by both the direct compression method and fluorescent RAA assay (both P values < 0.05). CONCLUSIONS Fluorescent RAA assay has a high sensitivity for detection of C. sinensis metacercariae in freshwater fish samples, and has no cross-reactions with M. orientalis, H. pumilio or C. formosanus metacercariae. Fluorescent RAA assay shows a higher accuracy for detection of C. sinensis infections in field-collected freshwater fish than the direct compression method.
Animals ; Clonorchis sinensis/genetics* ; Metacercariae/genetics* ; Recombinases ; Fresh Water ; Fishes ; DNA ; Fish Diseases/diagnosis*

Effects of gamma irradiation on the worm survival and chromosomal aberration of Clonorchis sinensis were studied. The metacercariae irradiated with various amounts of gamma radiation (ranging from 5 Gy to 50 Gy) were fed to rats, and the effects were compared with those of non-irradiated controls. Recovery rates of adult worms in irradiated groups were reduced gradually as increasing of the irradiation doses. No worm was recovered from rats which were fed with 50 Gy irradiated metacercariae. The chromosome number was 2n = 56 in all worms from all experimental groups. However, the groups irradiated with 20 Gy, 25 Gy or 30 Gy showed variations in the chromosome number, depending on different cells in the same individual. Radiation doses used in this study did not appear to induce chromosome aberrations, however, irradiation with 30 Gy showed slightly reduced chromosome size.
Animals ; Chromosome Aberrations/*radiation effects ; Clonorchis sinensis/*genetics/physiology/*radiation effects ; Dose-Response Relationship, Radiation ; Gamma Rays/*adverse effects ; Rats

The nitric oxide (NO) formation and intrinsic nitrosation may be involved in the possible mechanisms of liver fluke-associated carcinogenesis. We still do not know much about the responses of inducible NO synthase (iNOS) induced by Clonorchis sinensis infection. This study was conducted to explore the pathological lesions and iNOS expressions in the liver of mice with different infection intensity levels of C. sinensis. Extensive periductal inflammatory cell infiltration, bile duct hyperplasia, and fibrosis were commonly observed during the infection. The different pathological responses in liver tissues strongly correlated with the infection intensity of C. sinensis. Massive acute spotty necrosis occurred in the liver parenchyma after a severe infection. The iNOS activity in liver tissues increased, and iNOS-expressing cells with morphological differences were observed after a moderate or severe infection. The iNOS-expressing cells in liver tissues had multiple origins.
Animals ; Clonorchiasis/*enzymology/genetics/parasitology/*pathology ; Clonorchis sinensis/*physiology ; Female ; Humans ; Liver/*enzymology/parasitology/pathology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide Synthase Type II/*genetics/metabolism

Objective: This study aims to investigate the infection of Method: Infection of the definitive human host and intermediate fish host by Results: In 2016-2020, the average population infection rate of Hunan was 1.38%, while in Tongdao County the rate was up to 26.90%, and the highest fish infection rate was detected in Qiyang County (99.44% in the dorsal fin of Conclusion The systematically study of
Animals ; Cat Diseases/parasitology* ; Cats ; China/epidemiology* ; Clonorchiasis/veterinary* ; Clonorchis sinensis/genetics* ; Dog Diseases/parasitology* ; Dogs ; Fish Diseases/parasitology* ; Fishes ; Humans ; Incidence ; Prevalence ; Species Specificity

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-senstive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.
Adenosine Triphosphate/metabolism ; Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Clonorchis sinensis/*genetics/metabolism ; Helminth Proteins/*genetics/metabolism ; Human ; Molecular Sequence Data ; Potassium Channels, Inwardly Rectifying/*genetics/metabolism ; RNA, Helminth/chemistry/genetics ; Sequence Alignment ; Support, Non-U.S. Gov't

The evolutionary course of the CsRn1 long-terminal-repeat (LTR) retrotransposon was predicted by conducting a phylogenetic analysis with its paralog LTR sequences. Based on the clustering patterns in the phylogenetic tree, multiple CsRn1 copies could be grouped into four subsets, which were shown to have different integration times. Their differential sequence divergences and heterogeneous integration patterns strongly suggested that these subsets appeared sequentially in the genome of C. sinensis. Members of recently expanding subset showed the lowest level of divergence in their LTR and reverse transcriptase gene sequences. They were also shown to be highly polymorphic among individual genomes of the trematode. The CsRn1 element exhibited a preference for repetitive, agenic chromosomal regions in terms of selecting integration targets. Our results suggested that CsRn1 might induce a considerable degree of intergenomic variation and, thereby, have influenced the evolution of the C. sinensis genome.
Animals ; Clonorchis sinensis/*genetics ; DNA, Helminth/analysis/genetics ; *Evolution, Molecular ; Gene Dosage ; *Genome ; Phylogeny ; Polymorphism, Genetic ; RNA-Directed DNA Polymerase ; Retroelements/*genetics ; Sequence Analysis, DNA ; Terminal Repeat Sequences/*genetics

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4+/-0.09degrees C and 85.9+/-0.08degrees C for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.
Animals ; Asia ; Clonorchis sinensis/*classification/*genetics/isolation & purification ; Feces/parasitology ; Humans ; Multiplex Polymerase Chain Reaction/*methods ; NADH Dehydrogenase/genetics ; Opisthorchis/*classification/*genetics/isolation & purification ; Parasitology/*methods ; Real-Time Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Transition Temperature ; Zygote

Chemotherapy of clonorchiasis with praziquantel (PZQ) is effective but about 15% of treated cases have been reported uncured. The present study investigated correlation of single nucleotide polymorphisms (SNPs) of the cytochrome P450 gene, CYP3A5 and cure of clonorchiasis. A total of 346 egg passing residents were subjected and treated by 3 doses of 25 mg/kg PZQ. Reexamination recognized 33 (9.5%) uncured and 313 cured. Numbers of eggs per gram of feces (EPGs) before treatment were significantly lower in the cured group than in the uncured group (2,011.2+/-3,600.0 vs 4,998.5+/-7,012.0, P<0.001). DNAs of the subjects were screened for SNPs at 7 locations of CYP3A5 using PCR. In the uncured group, the SNP frequencies at g.-20555G>A and g.27526C>T of CYP3A5 were 15.2% and 9.1% while those were 3.8% and 1.0%, respectively, in the cured group. The cure rate was significantly lower in the cases with SNP at g.27526C>T and EPGs> or =1,000. In conclusion, EPGs and SNPs of CYP3A5 are factors which influence cure of clonorchiasis by PZQ therapy. It is strongly suggested to recommend 2-day medication for individuals with high EPGs> or =1,000.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Animals ; Child ; Clonorchiasis/*drug therapy/*genetics/parasitology ; Clonorchis sinensis/isolation & purification/physiology ; Cytochrome P-450 CYP3A/*genetics ; Feces/parasitology ; Female ; Humans ; Male ; Middle Aged ; *Polymorphism, Single Nucleotide ; Praziquantel/*therapeutic use ; Treatment Outcome ; Young Adult

In the course of immunoscreening of Clonorchis sinensis cDNA library, a cDNA CsRP12 containing a tandem repeat was isolated. The cDNA CsRP12 encodes two putative peptides of open reading frames (ORFs) 1 and 2 (CsRP12-1 and -2). The repetitive region is composed of 15 repeats of 10 amino acids. Of the two putative peptides, CsRP12-1 was proline-rich and found to have homologues in several organisms. Recombinant proteins of the putative peptides were bacterially produced and purified by an affinity chromatography. Recombinant CsRP12-1 protein was recognized by sera of clonorchiasis patients and experimental rabbits, but recombinant CsRP12-2 was not. One of the putative peptide, CsRP12-1, is designated CsPRA, proline-rich antigen of C. sinensis. Both the C-termini of CsRP12-1 and -2 were bacterially produced and analysed to show no antigenicity. Recombinant CsPRA protein showed high sensitivity and specificity. In experimental rabbits, IgG antibodies to CsPRA was produced between 4 and 8 weeks after the infection and decreased thereafter over one year. These results indicate that CsPRA is equivalent to a natural protein and a useful antigenic protein for serodiagnosis of human clonorchiasis.
Amino Acid Sequence ; Animals ; Antigens, Helminth/*genetics/isolation & purification ; Base Sequence ; *Cloning, Molecular ; Clonorchis sinensis/genetics/*immunology ; DNA, Helminth ; Gene Library ; Human ; Molecular Sequence Data ; Rabbits ; Recombinant Proteins ; *Repetitive Sequences, Nucleic Acid ; Support, Non-U.S. Gov't