Low Divergence of Clonorchis sinensis in China Based on Multilocus Analysis.

Clonorchis sinensis, an ancient parasite that infects a number of piscivorous mammals, attracts significant public health interest due to zoonotic exposure risks in Asia. The available studies are insufficient to reflect the prevalence, geographic distribution, and intraspecific genetic diversity of C. sinensis in endemic areas. Here, a multilocus analysis based on eight genes (ITS1, act, tub, ef-1a, cox1, cox3, nad4 and nad5 [4.986 kb]) was employed to explore the intra-species genetic construction of C. sinensis in China. Two hundred and fifty-six C. sinensis isolates were obtained from environmental reservoirs from 17 provinces of China. A total of 254 recognized Multilocus Types (MSTs) showed high diversity among these isolates using multilocus analysis. The comparison analysis of nuclear and mitochondrial phylogeny supports separate clusters in a nuclear dendrogram. Genetic differentiation analysis of three clusters (A, B, and C) showed low divergence within populations. Most isolates from clusters B and C are geographically limited to central China, while cluster A is extraordinarily genetically diverse. Further genetic analyses between different geographic distributions, water bodies and hosts support the low population divergence. The latter haplotype analyses were consistent with the phylogenetic and genetic differentiation results. A recombination network based on concatenated sequences showed a concentrated linkage recombination population in cox1, cox3, nad4 and nad5, with spatial structuring in ITS1. Coupled with the history record and archaeological evidence of C. sinensis infection in mummified desiccated feces, these data point to an ancient origin of C. sinensis in China. In conclusion, we present a likely phylogenetic structure of the C. sinensis population in mainland China, highlighting its possible tendency for biogeographic expansion. Meanwhile, ITS1 was found to be an effective marker for tracking C. sinensis infection worldwide. Thus, the present study improves our understanding of the global epidemiology and evolution of C. sinensis.

Identification and characterization of paramyosin from cyst wall of metacercariae implicated protective efficacy against Clonorchis sinensis infection.

Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.

Identification and molecular characterization of a novel signaling molecule 14-3-3 epsilon in Clonorchis sinensis excretory/secretory products.

Increasing evidence shows that 14-3-3 proteins are involved in many biology events in addition to signal transduction. Extensive investigations on structural and biochemical features of these signaling molecules have implied their importance in the biological process. In the present study, we have identified and characterized the 14-3-3 epsilon (Cs14-3-3) in Clonorchis sinensis that causes human clonorchiasis. Recombinant protein was expressed in Escherichia coli (E. coli) and identified by MALDI-TOF/TOF. Immunoblot results revealed that Cs14-3-3 was a component of excretory/secretory products. Ligand blot assay indicated that 14-3-3 epsilon could bind C. sinensis MAPKAPK 2 in a nonphosphorylation-dependent manner. This protein could be detected at four stages of the life cycle by RT-PCR experiments and immunolocalization showed that Cs14-3-3 was extensively distributed in C. sinensis, especially at the outer surface and the sucker of adult worm and cyst wall of metacercaria. Taken together, 14-3-3 epsilon might play some roles in the development of the parasites. In addition, Cs14-3-3 epsilon should be addressed for the diagnostic value in C. sinensis infection in consideration of high sensitivity and specificity. As an immune stimulus, C. sinensis 14-3-3 epsilon was found to provoke a Th1/Th2 balanced immune response by inducing high levels of both IgG1 and IgG2a. Recombinant Cs14-3-3 conferred effective protection both in worm reduction rate and egg reduction rate, suggesting that the signaling molecule Cs14-3-3 was a promising vaccine candidate against C. sinensis infection.

Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis.

Cysteine proteases play essential roles in parasite physiology as well as in host-parasite interactions through their modulation of various biological and pathobiological events. In the present study, a full-length sequence encoding cysteine protease of Clonorchis sinensis (CsCP) was isolated from our adult cDNA library. The open reading frame contains 984 bp encoding 327 amino acids. The present amino acid sequence shared 68% identity with two known CsCP genes and 29-49% identity with that of other species. Bioinformatics analysis showed that conserved domains and characteristic amino acid residues of cysteine proteases were observed in this sequence. Real-time PCR experiments revealed that CsCP was consecutively transcribed in various developmental stages of the parasite, including adult worm, excysted juvenile, metacercaria and egg. Recombinant CsCP (rCsCP) could be probed by rat anti-CsCP serum, rabbit anti-excretory-secretory products (ESP) serum and serum from human infected with Clonorchis sinensis in Western blot. The result of immunolocalization showed that CsCP was mainly located in the oral sucker, excretory bladder and tegument of cercariae and metacercariae, as well as the intestine of adult worm. The rCsCP-based IgG and its isotypes were all detected in sera from human infected with C. sinensis by enzyme-linked immunosorbent assay, and the level of IgG1 is the highest. The receiver-operating characteristic (ROC) analysis was used to determine the most appropriate cut-off value that yielded the high sensitivity (86.96%) and specificity (70.42%). These results revealed that CsCP may play an important role in the biology of C. sinensis and could be a diagnostic candidate for clonorchiasis.

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis.

BACKGROUND: Clonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome. RESULTS: We combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines. CONCLUSIONS: This study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.

Molecular characterization of cathepsin B from Clonorchis sinensis excretory/secretory products and assessment of its potential for serodiagnosis of clonorchiasis.

BACKGROUND: Cathepsin cysteine proteases play multiple roles in the life cycle of parasites such as food uptake, immune invasion and pathogenesis, making them valuable targets for diagnostic assays, vaccines and drugs. The purpose of this study was to identify a cathepsin B of Clonorchis sinensis (CsCB) and to investigate its diagnostic value for human helminthiases. RESULTS: The predicted amino acid sequence of the cathepsin B of C. sinensis shared 63%, 52%, 50% identity with that of Schistosoma japonicum, Homo sapiens and Fasciola hepatica, respectively. Sequence encoding proenzyme of CsCB was overexpressed in Escherichia coli. Reverse transcription PCR experiments revealed that CsCB transcribed in both adult worm and metacercaria of C. sinensis. CsCB was identified as a C. sinensis excretory/secretory product by immunoblot assay, which was consistent with immunohistochemical localization showing that CsCB was especially expressed in the intestine of C. sinensis adults. Both ELISA and western blotting analysis showed recombinant CsCB could react with human sera from clonorchiasis and other helminthiases. CONCLUSIONS: Our findings revealed that secreted CsCB may play an important role in the biology of C. sinensis and could be a diagnostic candidate for helminthiases.

Clonorchis sinensis enolase: identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products.

Enolase plays a key role in energy metabolism and development of most organisms. We isolated a gene encoding enolase from Clonorchis sinensis (C. sinensis) adult cDNA library and expressed the recombinant protein in Escherichia coli. C. sinensis enolase (Csenolase) was identified as both an excretory/secretory product and a tegumental component of C. sinensis by western blot analysis. The transcriptional level of Csenolase was examined at adult worm, metacercaria, cercaria and egg of C. sinensis, and results showed that Csenolase is transcribed at the four life stages of C. sinensis while showing a significant higher expression level at the stage of adult worm. Immunohistochemical localization indicated that Csenolase was specifically deposited on the tegument of adult worm and cyst wall of metacercaria. Ligand blot assay revealed a specific characteristic of dose-dependent plasminogen-binding activity of Csenolase and kinetic parameters were explored using 2-phospho-D-glycerate (2-PGA) as the primary substrate by monitoring the conversion of nicotinamide-adenine dinucleotide (NADH) into nicotinamide adenine dinucleotide (NAD). In addition, Csenolase exhibited active enzyme activity in catalytic reactions while the anti-Csenolase serum inhibited the enzyme activity. In vitro incubation experiments revealed that Csenolase might play key roles in the growth of the parasites. In conclusion, Csenolase is an important glycolytic enzyme required for the development of C. sinensis, and may be a potential vaccine candidate and drug target against C. sinensis infection.

Molecular cloning and characterization of a novel ras-related protein (rap2) from Clonorchis sinensis.

Ras are key components of diverse signal transduction pathways and play important roles in growth and development. To know about growth regulation in Clonorchis sinensis, we have identified a full-length sequence encoding a ras-related protein (rap2) from our adult cDNA library. The open reading frame contains 561 bp encoding 186 amino acids. The hypothetical amino acid sequence shared high identities with rap2 proteins from Schistosoma japonicum and Homo sapiens. Conserved domains of small guanosine triphosphate-binding proteins and characteristic amino acid residues of rap2 proteins were observed in this sequence. Reverse transcription polymerase chain reaction experiments revealed that rap2 transcribed in adult worm, metacercaria, and eggs of C. sinensis. Recombinant rap2 protein was expressed and purified from Escherichia coli. rap2 could be probed by C. sinensis-infected rat serum in western blotting experiment. By immunohistochemistry, rap2 was localized on the tegument of adult worm and metacercaria of C. sinensis. This fundamental study might contribute to further researches in signaling systems that are related to growth control and development of C. sinensis and other parasites.

Development and immunogenicity of a recombinant pseudorabies virus expressing Sj26GST and SjFABP from Schistosoma japonicum.

Recombinant pseudorabies virus (PRV) Bartha-K61 vaccine strains expressing Schistosoma japonicum 26kDa glutathione S-transferase (Sj26GST) and fatty acid binding protein (SjFABP), designated as rPRV/Sj26GST, rPRV/SjFABP and rPRV/Sj26GST-SjFABP, were constructed and evaluated for their ability to protect mice and sheep against S. japonicum challenge. Animals were given 2 intramuscular immunizations 3 weeks apart, and challenged with S. japonicum cercariae 4 weeks later. All mice vaccinated with recombinant virus developed specific anti-SWAP (soluble worm antigen preparation) antibody, splenocyte proliferative response and production of IFN-gamma and IL-2. Injection of rPRV/Sj26GST-SjFABP significantly increased levels of antibody, splenocyte proliferative response and production of IFN-gamma, compared with rPRV/Sj26GST and rPRV/SjFABP. These recombinant viruses have been shown to be safe for sheep. Challenge experiments showed worms and egg burdens were significantly reduced in animals immunized with recombinant PRVs. Most importantly, rPRV/Sj26GST-SjFABP dramatically enhanced protection with worm reduction and hepatic reduction of 39.3% and 45.5% respectively in mice, and 48.5% and 51.2% in sheep, while rPRV/Sj26GST and rPRV/SjFABP provided corresponding protection of only up to 23.7% and 27.2% in mice, and 29.0% and 35.5% in sheep. These results indicate that the multivalent vaccine for S. japonicum can produce significant specific immunity and protection, and that PRV Bartha-K61 is an effective live vector for an animal schistosomiasis japonica vaccine.

IL-18 enhances protective effect in mice immunized with a Schistosoma japonicum FABP DNA vaccine.

Two recombinant plasmids, pVAX/SjFABP and pVAX/mIL-18 containing Schistosoma japonicum 14 kDa fatty acid binding protein (SjFABP) and murine IL-18, were constructed and evaluated for their ability to induce immune responses and to protect against S. japonicum challenge in mice. Mice were intramuscularly immunized twice at three-weekly intervals, and challenged with S. japonicum cercariae at 4 weeks after the last vaccination. All animals vaccinated with pVAX/SjFABP alone or plus pVAX/mIL-18 developed specific anti-SWAP ELISA antibody and T lymphocyte proliferation. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-gamma and IL-2 compared with pVAX/SjFABP alone, indicating that IL-18 enhances the Th1-dominant immune response. The challenge experiment showed that co-injection of plasmid encoding IL-18 significantly enhances protective effect against S. japonicum infection, as demonstrated by worm reduction rates and the hepatic egg reduction rates 45 days post-challenge. These results indicated that IL-18 may become a novel vaccine adjuvant for development of vaccines against schistosomiasis.

Protection in mice immunized with a heterologous prime-boost regime using DNA and recombinant pseudorabies expressing TgSAG1 against Toxoplasma gondii challenge.

An effective vaccine of animals can block transmission of Toxoplasma gondii to humans. In this study, mice have been protected against lethal T. gondii challenge by a prime-boost vaccination strategy using DNA vaccine pVAX/TgSAG1 and recombinant pseudorabies virus rPRV/TgSAG1, both expressing the major immunodominant surface antigen of T. gondii (TgSAG1). High levels of splenocyte proliferative responses and significant levels of IFN-gamma resulted, with strong cytotoxic T lymphocyte (CTL) responses in vitro. After lethal challenge, prime-boost vaccinated mice showed an increased survival time (15.4+/-5.0 days) and a 40% survival rate compared with controls who all died within 11 days of challenge. Results of the present study indicated that this novel immunization strategy is useful in enhancing immune protection in mice against lethal T. gondii infection, which would provide foundation for the development of effective vaccines against T. gondii.

A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice.

The major immunodominant surface antigen 1 (TgSAG1) of invasive tachyzoites is a vaccine candidate antigen for Toxoplasma gondii. In this study, we developed a recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV/SAG1) based on the PRV vaccine strain Bartha K-61 by homologous recombination, in which partial PK and gG genes were deleted. The growth assay of rPRV/SAG1 showed that the recombinant virus can replicate in vitro as efficiently as PRV Bartha K-61, demonstrating that insertion of the TgSAG1 gene in the PK and gG locus of PRV does not affect the replication of PRV. All mice vaccinated with rPRV/SAG1 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-gamma and IL-2 production. And the immunization of mice with rPRV/SAG1 elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrate that rPRV/SAG1 could induce significant humoral and cellular Th1 immune responses. Moreover, rPVR/SAG1 immunization induced partial protection (60%) against a lethal challenge with the highly virulent T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. These results suggest that expression of protective antigens of T. gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against both animal toxoplasmosis and pseudorabies.

Enhancement by IL-18 of the protective effect of a Schistosoma japonicum 26kDa GST plasmid DNA vaccine in mice.

Two recombinant plasmids pVAX/Sj26GST and pVAX/mIL-18 containing Schistosoma japonicum 26kDa GST and murine IL-18 were evaluated for their ability to protect mice against S. japonicum challenge. Mice were given 2 intramuscular immunizations 3 weeks apart, and challenged with S. japonicum cercariae 4 weeks later. Adult worm and egg burdens were determined 48 days post-challenge. All animals vaccinated with pVAX/Sj26GST alone or with pVAX/mIL-18 developed specific anti-SWAP (soluble worm antigen preparation) ELISA antibody and splenocyte proliferation response. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-gamma and IL-12, indicating that IL-18 enhances the Th1-dominant immune response. Challenge experiments showed that worms were reduced in the pVAX/Sj26GST group by 30.1% and by 49.4% in animals given pVAX/mIL-18 additionally. Corresponding hepatic and fecal egg reductions were 44.8% and 53.0%, and 50.6% and 56.6%, respectively. These results indicate that IL-18 may be an effective adjuvant for a schistosomiasis vaccine.