Clonorchis sinensis and clonorchiasis, an update

Clonorchis sinensis is the most common human liver fluke in East Asia. Several studies proved its carcinogenesis in humans and it was reclassified as a group 1 biological carcinogen in 2009. It is still actively transmitted in endemic areas of Korea, China, Russia, and Vietnam. Currently it is estimated that more than 200 million people are at risk of infection, 15-20 million people are infected and 1.5-2 million show symptoms or complications. Several molecules and genes of the fluke have been identified and characterized. Studies on its oncogenesis and omics-based findings have been especially encouraging. Diagnosis of its infection depends mainly on detection of eggs in feces but other methods have been developed. ELISA using crude extract antigen is now popular for its diagnosis. Diagnosis by detecting DNAs from eggs in feces has been developed using PCR, real-time PCR, and LAMP, which have been found sensitive and specific. Imaging diagnosis has been studied in depth and is widely used. Any evidence of clonorchiasis, such as eggs, DNAs, or images, may lead to recommendations of chemotherapy in endemic areas. Praziquantel is the major chemotherapeutic agent for clonorchiasis and recently tribendimidine was found effective and is now under investigation as a promising chemotherapeutic alternative. Sustainable control programs which include mass chemotherapy with praziquantel and education for prevention of re-infection may reduce its morbidity and eliminate its infections in endemic areas.     

Development of resistance to reinfection by Clonorchis sinensis in rats

We investigated the induction of resistance to Clonorchis sinensis infection by prior infection in rat and hamster models. Animals were challenged with C. sinensis metacercariae, then treated with praziquantel and reinfected. Worm recovery rate in reinfected animals was used to estimate resistance to reinfection. The determined resistance rates to reinfection in rats and hamsters were 97.7% and 10.3%, respectively. In rats, cure from the primary infection of C. sinensis increased resistant to reinfection, and the greater the worm burden and the longer the duration of primary infection, the higher was the resistance rate. For primary infection doses of 10, 40 and 100 metacercariae per rat, the resistance rates were 87.4%, 93.8% and 98.4%, respectively. The resistance rates in rats after 2 or 8-week primary infection were 78.7% and 95.3%, respectively. All worms recovered from reinfected rats were immature. When cured rats were administered with methylprednisolone, resistance to reinfection became impaired. These findings indicate that rats develop a high degree of resistance to reinfection by C. sinensis after cure. The growths and maturations of reinfected worms were also impaired.     

Genomics and molecular genetics of Clonorchis sinensis: current status and perspectives

Clonorchiasis caused by Clonorchis sinensis is an important foodborne parasitosis of humans and animals, and is predominantly a hepatobiliary disease. Globally, nearly 35 million people were infected with C. sinensis, with approximately 15 million being in China. Patients would chronically present fatigue, jaundice, abdominal discomfort, along with the increased risk of developing into a form of cholangiocarcinoma that is fatal to humans. Treatment of clonorchiasis by praziquantel has been very successful, but this is dependent on early accurate diagnosis and correct species identification. The present article reviews the current status of knowledge in genomics and functional genomics of C. sinensis, and summarizes the main DNA-based techniques for the specific diagnosis of C. sinensis infection and studies of genetic variation in C. sinensis, and provides perspectives for future studies. The advances in genomics and molecular genetics of C. sinensis shed new sight on our understanding of population structure of C. sinensis as well as the prevention and control of clonorchiasis.     

Bile duct changes in rats reinfected with Clonorchis sinensis

This study describes an evaluation of the sonographic, cholangiographic, pathological, and immunological findings, and the protective effect shown by rats reinfected with Clonorchis sinensis. Eight experimental rat groups were, namely, a normal control, a primary infection control, a reinfection I (reinfection 7 week after treatment following 3-week infection), a reinfection II (reinfection 2 week after treatment following 8-week infection), a reinfection III (exploration of the intrahepatic bile ducts 1 week after reinfection 4 week after treatment following 4-week infection), a superinfection, a secondary infection control, and an infection following immunization group. Sonographic and cholangiographic findings showed moderate or marked dilatation of the bile duct confluence in the primary infection control, reinfection II, and secondary infection control groups. Juvenile worms survived in the intrahepatic bile ducts 1 week after reinfection following treatment in the reinfection III group. It was concluded that reinfecting juvenile worms found during the first week following reinfection failed to survive or grow further. Anatomical, pathophysiological, or immunological changes may induce protection from reinfection in rats.     

Cyst formation, increased anti-inflammatory cytokines and expression of chemokines support for Clonorchis sinensis infection in FVB mice

To verify the hypothesis that different pathology of Clonorchis sinensis infection by mouse strains is determined by different responses of cytokines and chemokines, we compared those responses of FVB with those of BALB/c mice. All of FVB mice infected with 30 metacercariae of C. sinensis showed cystic dilatation in the liver, whereas infected BALB/c mice did not. Mature worms were recovered from 19 of 20 liver sections of FVB mice while only one of 20 sections of BALB/c mice revealed a mature worm. In both strains the proportion of CD4⁺ T cells was lower in C. sinensis-infected than in the uninfected group. However, the proportion of CD8⁺ T cells was elevated in C. sinensis-infected from both strains compared to uninfected mice. The Th2-associated anti-inflammatory cytokines such as IL-4, IL-5 and IL-13, IL-10 and TGF-β, were significantly more produced by the lymphocytes of FVB than by those of BALB/c mice. Especially, the 2 anti-inflammatory cytokines, IL-10 and TGF-β, were presumably related with susceptibility and the development of worms in the liver. C. sinensis infected FVB mice also produced more chemokines such as RANTES and MIP-1α in the liver lymphocytes than BALB/c mice. In conclusion, the FVB mice provide the favorable niche for C. sinensis by cyst formation in the bile duct, increased production of Th2-associated anti-inflammatory cytokines and upregulation of chemokines.     

Collection of Clonorchis sinensis adult worms from infected humans after praziquantel treatment

A cohort was established for evaluation of cancer risk factors in Sancheong-gun, Gyeongsangnam-do, Korea. As one of the cohort studies, stools of 947 residents (403 males and 544 females, age range: 29-86 years) were screened for Clonorchis sinensis eggs using both Kato-Katz method and formalin-ether sedimentation technique. The overall egg positive rate of C. sinensis was 37.7% and individual EPG (eggs per gram of feces) counts ranged from 24 to 28,800. Eight egg positive residents voluntarily joined a process of collection of the passed worms after praziquantel treatment. A total of 158 worms were recovered from 5 of the 8 treated persons, ranged from 3 to 108 in each individual. The worms were 15-20 mm x 2-3 mm in size, and showed brown-pigmented, red, or white body colors. This is the first collection record of C. sinensis adult worms from humans through anthelmintic treatment and purgation. The adult worms of C. sinensis may be paralyzed by praziquantel and then discharged passively through bile flow in the bile duct and by peristaltic movement of the bowel.     

Antigenic profile and localization of Clonorchis sinensis proteins in the course of infection.

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunohistochemical staining. In the early stage of infection until 12 weeks post-infection (PI), antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI, antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection.     

Organ-specific antigens of Clonorchis sinensis

This study was carried out to find out specific proteins from different organs of Clonorchis sinensis. Crude extract, organ-specific and excretory-secretory (ES) proteins were analyzed by immunoblot with infected human sera. The bands of 7- and 17-kDa were main component of intestinal fluid and ES protein and commonly found in all organspecific proteins. The 17-kDa protein was observed from ES antigen, intestinal fluid, eggs and sperms, 26- and 28-kDa proteins were from the uterus, vitellaria, and ovary, and 34-, 37-, 43- and 50-kDa proteins were mainly from the testis and sperms. Serum of mice immunized with sperms reacted to the 50-kDa protein by immunoblotting and immunohistochemical staining showed a positive reaction at the seminal receptacle and seminiferous tubule. The present results show that the 7-kDa protein is a common antigen of every part or organ of C. sinensis, but different organs express their specific antigenic protein bands.     

Clonorchis sinensis: development and evaluation of a nested polymerase chain reaction (PCR) assay

Clonorchis sinensis is a fish-borne trematode endemic to East Asia, which infects over 35 million people globally. In the study described here, we developed a nested polymerase chain reaction (PCR) method for the specific and reliable detection of C. sinensis. The primers designed from the nucleotide sequence data derived in this study were evaluated for their specificity and sensitivity for the detection of C. sinensis. The specific amplification products were obtained only with C. sinensis and no amplifications occurred with the DNA of closely related trematodes including Opisthorchis viverrini demonstrating the specificity of the assay. The novel PCR method described here will be useful for the quarantine of fishery products and evaluation of transmission status of clonorchiasis in the endemic areas.     

Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was found to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was estimated to be 110 kDa by Superose 6 HR 10/30 gel filtration. The purified antigen strongly reacted with the human sera of clonorchiasis. The hyperimmune sera of BALB/c mice immunized against the 8 kDa protein were reacted with both the crude extract and the excretory-secretory product of adult worms, but not with the metacercarial extract. Immunohistochemical staining demonstrated that the protein was distributed to the tegument and subtegumental cells and also to the seminal receptacle. The present findings suggest that the 8 kDa protein is a partition of the multicomplex protein originating from various organs of adult C. sinensis, and that it is composed of several 7 and 8 kDa proteins.     

Infection Status of Freshwater Fish with Metacercariae of Clonorchis sinensis in Korea

This study investigated freshwater fish for their current infection status with metacercariae of Clonorchis sinensis in Korea. Twenty-one species of freshwater fish (n = 677) were collected from 34 regions nationwidely from February 2007 to June 2008. They were individually examined by digestion technique. Eight species of freshwater fish from 17 different regions were recognized positive for the metacercariae of C. sinensis. The positive rates (range of metacercariae number per fish) of fish by the species were as follows: 48% (1-1,142) in Pseudorasbora parva, 60% (1-412) in Pungtungia herzi, 15.7% (1-23) in Pseudogobio esocinus, 29% (1-7) in Acheilognathus intermedia, 21% (1-4) in Odontobutis interrupta, 33% (1-6) in Zacco temmincki, 3.6% (1-4) in Zacco platypus, and 26.3% (1) in Hemibarbus labeo. The two species, P. parva and P. herzi, are able to be the index fish for estimation of C. sinensis transmission in a certain locality. Still several species of freshwater fish are briskly transmitting C. sinensis infection in many riverside areas of southern Korea.     

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis

Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.     

Chromosomes of the liver fluke, Clonorchis sinensis

A karyological study was carried out in order to compared the chromosome numbers, chromosome morphologies and karyotypes of the oriental liver fluke, Clonorchis sinensis (Trematoda: Opisthorchiidae), collected from Korea and China. Chromosome preparations were made by means of air-drying method. The chromosome number was 2n = 56 in both Korean and Chinese flukes, and chromosomes were divided into two groups based on this size; consisting of 8 pairs of large and 20 pairs of small chromosomes. However, the karyotypes showed some differences between Korean and Chinese flukes. The karyotype of liver flukes from Korea consisted of three metacentric pairs, one meta-/submetacentric pair, 16 submetacentric pairs and eight subtelocentric pairs of chromosomes. On the other hand, liver flukes from China consisted of two metacentric pairs, two meta-/submetacentric pairs, 16 submetacentric pairs and eight subtelocentric pairs of chromosomes.     

Partial characterization of a 17 kDa protein of Clonorchis sinensis

A 17 kDa protein from Clonorchis sinensis adults was purified by a procedure including Sephacryl S-200 HR gel filtration and Q-Sepharose anion exchange chromatography. The protein was proved to be a cysteine protease as it showed hydrolytic activity toward Cbz-Phe-Arg-AMC in the presence of dithiothreitol and was inhibited by specific inhibitors such as iodoacetic acid or trans epoxy-succinly-L-leucyl-amido(4-guanidino) butane. The polyclonal antibody raised against the protein reacted to 17 kDa proteins of trematodes such as Paragonimus westermani, Fasciola hepatica, Opisthorchis viverrini, Gymnophalloides seoi, and Metagonimus yokogawai. The antibody recognized the 17 kDa and 16 kDa cysteine proteases purified from C. sinensis, P. westermani, and G. seoi as well. These results suggest that the 17 kDa protein may be a cysteine protease commonly present in trematodes.     

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn²⁺ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.     

Effects of gamma-irradiation on the infectivity and chromosome aberration of Clonorchis sinensis

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.     

Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-sensitive 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.     

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1 pg of purified genomic DNA, 5 EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent.     

Changing patterns of serum and bile antibodies in re-infected rats with Clonorchis sinensis

Rats develop strong resistance to re-infection and super-infection by Clonorchis sinensis. The present study investigated the antibodies present in the sera and bile juice of rats that were primary infected and re-infected with C. sinensis. The serum level of specific IgG antibodies, which were elevated 2 wk of the primary infection, peaked at 4 wk and subsequently remained unchanged even during re-infection. The total IgE level in serum increased slowly from 388 ng / ml to 3,426 ng / ml beginning 2 wk after the primary infection, and remained high up to 8 wk but dropped to a normal level (259 ng / ml) after treatment. In resistant re-infected rats, the serum IgE level increased rapidly and peaked within 1 wk, whereas no increase was observed in immunosuppressed rats. The serum level of specific IgA antibodies was elevated beginning 1 wk after infection, and decreased 4 wk after treatment. The total bile IgA level unchanged during the primary infection but increased in treated and re-infected rats. The elevated levels of serum IgE and bile IgA indicate that these immunoglobulins may be correlated with the development of resistance to re-infection by C. sinensis in rats.     

A new faecal antigen detection system for Strongyloides venezuelensis diagnosis in immunosuppressed rats

This study was performed with the objective of developing innovative procedures for the diagnosis of strongyloidiasis. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect coproantigen in the faecal samples of normal and of immunosuppressed rats using an anti-L3 polyclonal antibody produced in rabbits. Analysis revealed the kinetics of egg shedding in the non-immunosuppressed and immunosuppressed rats infected with S. venezuelensis. Further analysis verified the ability of the immune serum to detect L3 antigens in faecal samples from infected animals. The number of eggs recovered in the faeces at 8 days p.i was significantly higher for both groups. Immunosuppressed animals eliminated increased quantities of eggs. The immune serum was able to detect 0.39 μg/ml of L3 antigens. The antigen recognition in the immunosuppressed group was anticipated on the 8th day p.i. In conclusion, these results may represent a first step in the development of a rapid coproantigen detection kit for strongyloidiasis.