Immunodiagnosis of clonorchiasis using a recombinant antigen

A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a β-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.     

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.     

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.     

Excretory-secretory antigen is better than crude antigen for the serodiagnosis of clonorchiasis by ELISA

Although stool examination is the standard diagnostic method of clonorchiasis, serodiagnosis by ELISA using crude antigen is now widely used because of its convenience. However, ELISA diagnosis still suffers from cross-reactions, and therefore there is a need to improve the present conventional ELISA. The present study was undertaken to evaluate the diagnostic value of ELISA using excretory-secretory antigen (ESA) instead of crude antigen (CA) of Clonorchis sinensis. The diagnostic sensitivity of ELISA using excretory-secretory antigen was 92.5%, which was higher than that of ELISA using crude Clonorchis sinensis antigen (88.2%). In addition, the specificity of excretory-secretory antigen was found 93.1% while that of crude antigen was 87.8%. In summary, Clonorchis sinensis ESA was found to be a better serodiagnostic antigen than CA for ELISA.     

Ac-SAA-1, an immunodominant 16 kDa surface-associated antigen of infective larvae and adults of Ancylostoma caninum

A cDNA encoding a surface-associated antigen was cloned from an Ancylostoma caninum infective larvae (L(3)) cDNA library by immunoscreening with pooled human immune sera. The sera were obtained from individuals living in an Ancylostoma duodenale hookworm-endemic region of China, who had light intensity infections and high antibody titers against A. caninum L(3). Ancylostoma caninum surface-associated antigen-1 is encoded by an 843 bp mRNA with a predicted open reading frame of 162 amino acids. Recombinant Ancylostoma caninum surface-associated antigen-1 was expressed in Escherichia coli and used to prepare a specific antiserum. A Western blot with anti-Ancylostoma caninum surface-associated antigen-1 specific antiserum showed that native Ancylostoma caninum surface-associated antigen-1 protein is expressed by both L(3) and adult hookworms; RT-PCR confirmed that the mRNA is transcribed in both stages. In adult hookworms, the protein localised to the basal layer of the cuticle and hypodermis of adult worms. Serological analysis determined that recombinant Ancylostoma caninum surface-associated antigen-1 protein is recognised by 61% of human sera from a Necator americanus hookworm endemic area in China, indicating the antigen is immunodominant. Anti-Ancylostoma caninum surface-associated antigen-1 antiserum partially inhibited (46.7%) invasion of hookworm L(3) into dog skin in vitro. Together these results suggest that Ancylostoma caninum surface-associated antigen-1 offers promise as a protective vaccine antigen.     

Molecular cloning and characterization of an antigenic protein with a repeating region from Clonorchis sinensis

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.     

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.     

Coproantigen capture ELISA for detection of Clonorchis sinensis infection in experimentally infected rats

The present study investigated the diagnostic value of an ELISA for the detection of Clonorchis sinensis antigen in the feces of experimentally infected rats. A mouse polyclonal IgG antibody against adult C. sinensis crude antigen (CsAg) was used to capture the C. sinensis coproantigen. The detection limit for pure CsAg was 20 ng/ml in sample buffer and 40 ng/ml in uninfected fecal extract. The test was evaluated using a follow-up of five groups of rats experimentally infected with 100, 50, 10, 5 and 1 metacercariae of C. sinensis and an uninfected control group. Coproantigen was detected in all infected groups of rats from 2 weeks of infection, whereas fecal eggs were not observed until 3 weeks of infection. As the infection period progressed, the fecal CsAg concentration increased in all groups of infected rats, even those infected with a single metacercaria. The fecal CsAg concentration was correlated positively with fecal egg counts and worm burden. This coproantigen capture ELISA is highly sensitive for the detection of CsAg in rat feces, and with further development, should be useful for mass screening of human subjects in clonorchiasis-endemic areas.     

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.     

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.     

In Vitro Maintenance of Clonorchis sinensis Adult Worms

Clonorchis sinensis is a biological carcinogen inducing human cholangiocarcinoma, and clonorchiasis is one of the important endemic infectious diseases in East Asia. The present study investigated survival longevity of C. sinensis adult worms in various in vitro conditions to find the best way of keeping the worms longer. The worms were maintained in 0.85% NaCl, 1×PBS, 1×Locke''s solution, RPMI-1640, DMEM, and IMDM media, and in 1×Locke''s solution with different supplements. All of the worms died within 3 and 7 days in 0.85% NaCl and 1×PBS, respectively, but survived up to 57 days in 1×Locke''s solution. The worms lived for 106 days in DMEM, and 114 days in both RPMI-1640 and IMDM media. The survival rate in RPMI-1640 medium was the highest (50%) compared to that in DMEM (20±10%) and in IMDM (33.3±25.2%) after 3 months. The 1×Locke''s solution with 0.005% bovine bile supplement showed increased duration of maximum survival from 42 days to 70 days. Higher concentration of bile supplements than 0.005% or addition of glucose were disadvantageous for the worm survival. The worms died rapidly in solutions containing L-aspartic acid, L-glutamic acid, and adenine compared to L-arginine, L-serine, and L-tryptophan. In conclusion, the 1×Locke''s solution best supports the worms alive among inorganic solutions for 57 days, and the RPMI-1640 medium maintains living C. sinensis adults better and longer up to 114 days in vitro than other media.     

Nitration and chlorination of folic acid by peroxynitrite and hypochlorous acid,and the selective binding of 10-nitro-folate to folate receptor beta

Peptides pore-forming in cell membrane have been identified from a wide range of animals. A putative pore-forming peptide deduced from a cDNA clone of Clonorchis sinensis (clonorin) was predicted to consist of four amphipathic alpha-helices. Clonorin contained six invariably conserved cysteine residues, identified to form three disulfide bonds. These predicted structural features are highly homologous with pore-forming peptides, the amoebapores. Recombinant clonorin showed hemolytic activity toward rabbit erythrocytes. The hemolytic activity of C. sinensis extract increased dose-dependently and was inhibited by anti-clonorin immune sera. The clonorin was expressed developmentally in juvenile and adult flukes and localized in the intestinal epithelium of adult flukes. It is proposed that, through lysing host cellular components, clonorin could enhance proteolytic digestion in the intestine of C. sinensis.     

Molecular Cloning and Characterization of a Paramyosin from Clonorchis sinensis

Paramyosin is a myofibrillar protein present in helminth parasites and plays multifunctional roles in host-parasite interactions. In this study, we identified the gene encoding paramyosin of Clonorchis sinensis (CsPmy) and characterized biochemical and immunological properties of its recombinant protein. CsPmy showed a high level of sequence identity with paramyosin from other helminth parasites. Recombinant CsPmy (rCsPmy) expressed in bacteria had an approximate molecular weight of 100 kDa and bound both human collagen and complement 9. The protein was constitutively expressed in various developmental stages of the parasite. Imunofluorescence analysis revealed that CsPmy was mainly localized in the tegument, subtegumental muscles, and the muscle layer surrounding the intestine of the parasite. The rCsPmy showed high levels of positive reactions (74.6%, 56/75) against sera from patients with clonorchiasis. Immunization of experimental rats with rCsPmy evoked high levels of IgG production. These results collectively suggest that CsPmy is a multifunctional protein that not only contributes to the muscle layer structure but also to non-muscular functions in host-parasite interactions. Successful induction of host IgG production also suggests that CsPmy can be applied as a diagnostic antigen and/or vaccine candidate for clonorchiasis.     

Clonorchis sinensis: molecular cloning and functional expression of novel cytosolic malate dehydrogenase

The NAD-dependent cytosolic malate dehydrogenase (cMDH, EC 1.1.1.37) plays a pivotal role in the malate-aspartate shuttle pathway that operates in a metabolic coordination between cytosol and mitochondria, and thus is crucial for the survival and pathogenicity of the parasite. In the high throughput sequencing of the cDNA library constructed from the adult stage of Clonorchis sinensis, a cDNA clone containing 1152bp insert was identified to encode a putative peptide of 329 amino acids possessing more than 50% amino acid sequence identities with the cMDHs from other organisms such as fish, plant, and mammal. But low sequence similarities have been found between this cMDH and mitochondrial malate dehydrogenase as well as glyoxysomal malate dehydrogenase from other organisms. Northern blot analysis showed the size of the C. sinensis cMDH mRNA was 1.2 kb. The cMDH was expressed in Escherichia coli M15 as a His-tag fusion protein and purified by BD TALON metal affinity column. The recombinant cMDH showed high MDH activity of 241 U mg(-1), without lactate dehydrogenase and NADP(H) selectivity. It provides a model for the structure, function analysis, and drug screening on cMDH.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

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.     

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.     

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.