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Clonorchiasis is a serious food borne parasitic disease caused by clonorchis sinensis that is transmitted via snails to freshwater fish, and then to humans and other mammals. Based on the life cycle clonorchis sinensis, we present a mathematical model of clonorchiasis with three time delays which represent the maturation period of metacercaria developing adult flukes within human host, the period of eggs developing cercariae within snail host and the period of cercariae developing metacercaria within fish host.The basic reproduction number R 0 for this model is introduced. We establish a threshold type result on the global dynamics in terms of R 0 . More precisely, the disease-free equilibrium is globally attractive if R 0 1 ; while the disease is uniformly persistent if R 0 > 1 . Considering the above-mentioned periods can also be modelled by introducing the corresponding exposed classes, we also study the dynamical behavior of the corresponding model without delays. Numerically, we study the clonorchiasis transmission case in Guangxi Pronvince, China. Some control strategies are provided to prevent clonorchiasis spread in Guangxi by numerical analysis.

The Chinese liver fluke, Clonorchis sinensis, causes the disease clonorchiasis, affecting ~35 million people in regions of China, Vietnam, Korea and the Russian Far East. Chronic clonorchiasis causes cholangitis and can induce a malignant cancer, called cholangiocarcinoma, in the biliary system. Control in endemic regions is challenging, and often relies largely on chemotherapy with one anthelmintic, called praziquantel. Routine treatment carries a significant risk of inducing resistance to this anthelmintic in the fluke, such that the discovery of new interventions is considered important. It is hoped that the use of molecular technologies will assist this endeavour by enabling the identification of drug or vaccine targets involved in crucial biological processes and/or pathways in the parasite. Although draft genomes of C. sinensis have been published, their assemblies are fragmented. In the present study, we tackle this genome fragmentation issue by utilising, in an integrated way, advanced (second- and third-generation) DNA sequencing and informatic approaches to build a high-quality reference genome for C. sinensis, with chromosome-level contiguity and curated gene models. This substantially-enhanced genome provides a resource that could accelerate fundamental and applied molecular investigations of C. sinensis, clonorchiasis and/or cholangiocarcinoma, and assist in the discovery of new interventions against what is a highly significant, but neglected disease-complex.

Objectives The aim of this study was to investigate the epidemic scope, epidemic pattern and degree of damage of clonorchiasis as well as to summarize the diagnosis and control effect of clonorchiasis in Shandong Province, China. Methods A retrospective study was conducted to summarize the research on the dynamic epidemiology and the prevention and treatment measures of clonorchiasis in Shandong Province, China from 1962 to 2015. Results After unremitting efforts and three nationwide surveys on the distribution and infection of human parasites, the infection rate of clonorchiasis decreased from 1.51% (1,500, 000) in the1960s to 0.16% in 1991, 0.04% in 2000 and 0.007% in 2015, respectively. The infection rate for villages with perennial water accumulation and rainy season water accumulation was higher than that of villages with rainy season water accumulation and non-water accumulation. The egg flotation in 70% potassium iodide solution method was applied to accurately test the specific proportion of C. sinensis eggs, which was 1.42. Using the modified Kato thick smear method, the detection rate of eggs was over 98%; hexachloroparaxylene yielded satisfactory therapeutic effects with slight adverse reactions, it is effective in the treatment of clonorchiasis. Conclusions After 50 years of active prevention and treatment, the disease has been successfully controlled in Shandong Province and had been used for reference in other endemic areas of clonorchiasis in China.

Liver flukes include Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis spp., Fascioloides magna, Gigantocotyle explanatum and Dicrocoelium spp. The two main species, F. hepatica and F. gigantica, are major parasites of livestock and infections result in huge economic losses. As with C. sinensis, Opisthorchis spp. and Dicrocoelium spp., they affect millions of people worldwide, causing severe health problems. Collectively, the group is referred to as the Food-Borne Trematodes and their true significance is now being more widely recognised. However, reports of resistance to triclabendazole (TCBZ), the most widely used anti-Fasciola drug, and to other current drugs are increasing. This is a worrying scenario. In this review, progress in understanding the mechanism(s) of resistance to TCBZ is discussed, focusing on tubulin mutations, altered drug uptake and changes in drug metabolism. There is much interest in the development of new drugs and drug combinations, the re-purposing of non-flukicidal drugs, and the development of new drug formulations and delivery systems; all this work will be reviewed. Sound farm management practices also need to be put in place, with effective treatment programmes, so that drugs can be used wisely and their efficacy conserved as much as is possible. This depends on reliable advice being given by veterinarians and other advisors. Accurate diagnosis and identification of drug-resistant fluke populations is central to effective control: to determine the actual extent of the problem and to determine how well or otherwise a treatment has worked; for research on establishing the mechanism of resistance (and identifying molecular markers of resistance); for informing treatment options; and for testing the efficacy of new drug candidates. Several diagnostic methods are available, but there are no recommended guidelines or standardised protocols in place and this is an issue that needs to be addressed., Graphical abstract Image 1, Highlights • Liver flukes are major parasites of humans and livestock and cause severe disease. • Resistance to triclabendazole and other flukicides is becoming a serious problem. • The mechanism of resistance to triclabendazole is not clear, but may have a polygenic basis. • Possible treatment and management strategies to deal with resistance are discussed. • Several diagnostic tests are available, but no recommended guidelines are in place.

The complete cox1 gene sequence was analysed for Metagonimus suifunensis from eight localities in the Russian southern Far East, and the level of variability was compared with that of Clonorchis sinensis from the same territory of Russia. These species belong to the superfamily Opisthorchioidea, have a similar distribution in the Russian southern Far East and share second intermediate and definitive hosts, but are distinguished by their first intermediate hosts belonging to different orders of caenogastropods. The data obtained showed that the nucleotide sequence variability of the M. suifunensis cox1 gene was significantly lower. This fact is considered in connection with a recent bottleneck passage for the M. suifunensis population, in contrast to C. sinensis, which could be due to the features of the Metagonimus life cycle under seasonal freezing temperatures, as well as historical geological and climatic changes in the Russian Far East. These factors could influence the microevolutionary processes and lead to a decrease in the level of variability in the M. suifunensis population. Based on the combination of genetic data and historical geo-processes in the region, the probable route of M. suifunensis expansion from the northern part of its current area in the Amur River basin to the southern territories of the Russian Far East is justified.

Food-borne trematodiases represent major neglected parasitic diseases. Trematodes of the family Opisthorchiidae including Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis are ranked eight on the global list of the 24 most prevalent food-borne parasites. Chronic O. felineus infection symptoms include precancerous lesions with the potential for malignancy. In recent decades, liver flukes of the family Opisthorchiidae have been extensively scientifically explored, however despite this the molecular mechanisms of O. felineus pathogenicity and its carcinogenic potential have not been studied. Opisthorchis felineus glutathione-dependent prostaglandin synthase (GST σ) is the major component of the excretory-secretory product of this liver fluke. We hypothesised that the activity of this enzyme is involved in the infection pathogenesis, including the formation of precancerous lesions. To test this hypothesis and to gain insights into the mechanisms of precancerous lesion formation, we (i) investigated whether excretory parasitic GST σ retains its enzymatic activity, (ii) tested resveratrol (RSV) as a possible inhibitor of this enzyme, and (iii) assessed biliary neoplasia and oxidative DNA damage as well as the expression of neoplasia and fibrogenesis marker genes after prolonged administration of RSV in a hamster model. RSV was found to inhibit GST σ enzymatic activity in a dose-dependent manner (R = 0.85, P 0.001; half-maximal effective dose (ED

Background Clonorchiasis has been known for over 2300 years in China and it is considered as one of the most notorious food-borne parasitic diseases. In 2004, a total number of 15 million people were infected with Clonorchis sinensis around the world, in which more than 85% people lived in China. This was the first epidemical study about Clonorchis sinensis metacercariae and Metorchis orientalis metacercariae infection in freshwater fishes in seven endemic regions of China. Methods A total number of 2909 samples were collected from year 2015–2017. The regions of epidemic area of C. sinensis and Metorchis orientalis included Jilin, Heilongjiang, Shanghai, Zhejiang, Anhui, Guangdong and Guangxi provinces of China. 16 fish species were examined. Encysted metacercariae of C. sinensis and Metorchis orientalis were examined with digestion technique. The chi-square test, the Fisher's exact test and the logistic regression were applied in this study. Results The infection rate of C. sinensis metacercariae was 3.10% (90/2904) and that of Metorchis orientalis metacercariae was 1.20% (34/2831). The infected freshwater fishes were sold in different locations including fish pond, farm produce market, supermarket and restaurant. The infection rates of C. sinensis metacercariae and Metorchis orientalis metacercariae in Pseudorasbora parva were the highest up to 25.00% (17/68) and 14.93% (10/67), respectively. Conclusions Fish species was the major influence factor to the infection rates of C. sinensis metacercariae and Metorchis orientalis metacercariae.

Tribendimidine (TBD) is a broad-spectrum anthelmintic drug that is also significantly effective in treating clonorchiasis. In this study, the altered metabolomes of Clonorchis sinensis (C. sinensis) in rats after TBD administration were quantified by using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) to explore the possible active sites of TBD against clonorchiasis through altered metabolites and metabolic pathway analysis, and the results are expected to provide a target for the future design of anti-Clonorchis sinensis drugs. The worm burden reduction rate and scanning electron microscopy demonstrated that praziquantel (PZQ, positive control drug) and TBD had significant effects on C. sinensis in rats after treatment at a single dose of 200 mg/kg for 24 h. For the MS-based metabolomic analysis, a total of 173 standard metabolites (126 amino acids, 10 phospholipids and 37 fatty acids) were utilized as a reference metabolite database for metabolome identification. In total, 32 amino acids, 71 phospholipids and 27 fatty acids were detected in the C. sinensis of each group. Among these metabolites, 10 amino acids were significantly decreased in both drug-treated groups. Four lysophosphatidyl cholines (LPCs), six lysophosphatidyl ethanolamines (LPEs) and one phosphatidyl inositol (PI) were significantly increased after treatment with TBD. There were no significant changes in fatty acids among the control group and the two drug-treated groups. The results indicated that TBD administration caused a decrease in amino acids involved in the metabolic pathways of energy consumption and an increase in lysophospholipids, which are the hydrolysis products of phospholipase2 (PLA2) in the phospholipid metabolic pathways. The increased lysophospholipid content can destroy the cell membrane, increase membrane permeability, and even cause exposure to internal antigens that can be attacked by host antibodies. Perhaps the destroyed membrane, the exposed internal antigens and the consumed energy are the cause of the damage and death of C. sinensis after TBD administration. This is an interesting problem that can be examined in future research.

Both Fasciola hepatica and Clonorchis sinensis are endemic in China, South Korea, Japan and other Southeast Asian countries. Reliable and sensitive diagnostic methods are needed for detecting their infections in humans and animals. Differential simplex and duplex polymerase chain reaction (PCR) methods were developed. The PCRs targeted the second internal transcribed spacer (its2) (408 bp) of F. hepatica, and NADH dehydrogenase subunit 2 gene (nad2) (527 bp) of C. sinensis. Both simplex PCRs detected as little as 2 pg genomic DNA in one microliter in a 25 μL PCR reaction system. The duplex PCR had similar detection limit as well, and detected as low as one egg in 200 mg feces. These methods were analytical specific with no amplification being observed from the gemonic DNA of Fasciolopsis buski, Haemonchus contortus, Ascaris ovis or Eimeri ahsata. Of 158 sheep fecal samples collected from various farms, four and one samples were PCR-positive for F. hepatica and C. sinensis, respectively. The duplex PCR method described here is time-saving and convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of F. hepatica and C. sinensis in endemic area.

A total of 71 virulence and immunomodulation-related transcripts (VIRs) of Fasciola hepatica have been previously proposed (Haçarız et al., 2015). In an attempt to further refine this cohort, an in silico meta analysis approach was carried out using publicly available sequence data of related liver flukes, Clonorchis sinensis and Opisthorchis viverrini. Data of both liver flukes were investigated in terms of sequential homology with data of non-parasitic organisms, pathogens and VIRs of F. hepatica, directional selection (Ka/Ks), and cytokine signaling relation (protein motif based). Some VIRs of F. hepatica [showing homology with immune receptors (for toll/interleukin-1, TGF-β or TNF-α), TGF-β, TNF-α, CD147, or relation with suppressors of cytokine signaling/IKBKE 1 or stimulation of TGF-β (through thrombospondin similarity)] were found to be orthologous with those of both C. sinensis and O. viverrini. The in silico analysis indicates that on the basis of genetic commonality, a total of 30 VIRs of F. hepatica are highlighted as of foremost importance in the parasite evasion strategy, through controlling of host immune system. Findings in this study could be important to further enhance our understanding of the parasitic mechanisms and develop effective control strategies against F. hepatica and other related parasites.

Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3 Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel β-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.

The Greater Mekong Subregion (GMS) is a trans-national region of the Mekong River basin in Southeast Asia comprising Cambodia, the People's Republic of China (specifically Yunnan Province and Guangxi Zhuang Autonomous Region), Lao People's Democratic Republic (Lao PDR), Myanmar, Thailand, and Vietnam home to more than 340 million people or almost 4.5% of the global population. Human liver fluke infections caused by Opisthorchis viverrini and Clonorchis sinensis have been major public health problems in this region for decades. Opisthorchiasis caused by O. viverrini is prevalent in Thailand, Lao PDR, Cambodia and central-southern Vietnam with more than 12 million people infected. Clonorchiasis caused by C. sinensis is endemic in northern Vietnam and Guangxi with estimated 3.5 -5 million infected. The infections can cause several liver and biliary diseases such as cholangitis, periductal fibrosis, gallstones, and cholangiocarcinoma (CCA), a fatal bile duct cancer. Key determinants of the geographical distribution differences of the two liver fluke species are snail species and geographic barriers. Main risk behaviour of the infections is the culture of eating raw fish "the raw attitude" of people in the GMS, especially the Tai/Dai/Thai/Laos ethnic groups, the major population in the GMS. Over the past 20 years, there is a big change in prevalence of the infections. Opisthorchiasis has long been endemic, particularly in northern and northeastern Thailand and Lao PDR with over 8-10 million cases estimated. However, after several rounds of national campaign against opisthorchiasis using integrated control approach in Thailand over the past three decades, the prevalence of O. viverrini infection has reduced from over 15% in 1996 to 2.2% in 2019. High prevalence of O. viverrini infection continues in Lao PDR and central Vietnam. Emerging high prevalence, up to a maximum of 47.5%, has been noted in Cambodia during the past 10 years possibly due to more studies being conducted rather than increasing prevalence. O. viverrini infection has now also been reported in Lower Myanmar in recent years. Clonorchiasis has been known in northern Vietnam and southern China for a long time. Several surveys have reported infections in Guangxi in the last 10 years, and until now liver fluke infected cases have not been reported in Yunnan. Overall, nowadays, there is a shift in high risk areas for GMS liver fluke infection from northeastern Thailand to Lao PDR, Cambodia, Vietnam, Myanmar, and Guangxi P.R. China. Urgent systematic disease mapping and integrated liver fluke control using One Health approaches should be implemented nationwide in GMS countries.

Fasciola spp., Opisthorchis spp. and Clonorchis sinensis are common liver flukes that can cause a variety of diseases, mainly cholangiocarcinoma induced by clonorchiasis and liver damage and associated pathology induced by fascioliasis. Because these trematodes are parasites of humans and domestic animals, they have greatly affected the economy of agricultural industries and public health worldwide. Due to the emergence of drug resistance and the living habits of flukes, among other reasons, a possibility of reinfection remains even when antiparasitic drugs are used. Therefore, developing a safe, efficient and cost-effective vaccine against trematodes is an important goal. Here, we briefly describe the progress in the development of vaccines against liver flukes. Related innovations may provide effective protection against these helminths and the diseases that they cause.

Cu/Zn-superoxide dismutase (SOD1) is an enzyme that disproportionates superoxide anion into hydrogen peroxide and molecular oxygen. The enzymatic activity of SOD1 requires the binding of copper and zinc ions and also the formation of a conserved intramolecular disulfide bond. In a eukaryotic cell, a copper chaperone for SOD1 (CCS) has been known to supply a copper ion and also introduce the disulfide bond into SOD1; however, a mechanism controlling the CCS-dependent activation of SOD1 remains obscure. Here, we characterized CCS isolated from a human liver fluke, Clonorchis sinensis, and found that an N-terminal domain of CCS was essential in supplying a copper ion in SOD1. Regardless of the presence and absence of the N-terminal domain, CCS was able to bind a cuprous ion at the CxC motif of its C-terminal domain with quite high affinity (Kd~10-17). The copper-bound form of full-length CCS successfully activated C. sinensis SOD1, but that of CCS lacking the N-terminal domain did not. Nonetheless, the N-terminally truncated CCS with the bound copper ion was found to correctly introduce the disulfide bond into SOD1. Based upon these results, we propose that the N-terminal domain of CCS has roles in the release of the copper ion bound at the C-terminal domain of CCS to SOD1.

Liver flukes Fasciola hepatica, Opisthorchis viverrini and Clonorchis sinensis are causing agents of liver and hepatobiliary diseases. A remarkable difference between such worms is the fact that O. viverrini and C. sinensis are carcinogenic organisms whereas F. hepatica is not carcinogenic. The release of secretory factors by carcinogenic flukes seems to contribute to cancer development however if some of these target the host cell nuclei is unknown. We investigated the existence of O. viverrini and C. sinensis secretory proteins that target the nucleus of host cells and compared these with the corresponding proteins predicted in F. hepatica. Here we applied an algorithm composed by in silico approaches that screened and analyzed the potential genes predicted from genomes of liver flukes. We found 31 and 22 secretory proteins that target the nucleus of host cells in O. viverrini and C. sinensis, respectively, and that have no homologs in F. hepatica. These polypeptides have enriched the transcription initiation process and nucleic acid binding in O. viverrini and C. sinensis, respectively. In addition, other 11 secretory proteins of O. viverrini and C. sinensis, that target the nucleus of host cells, had F. hepatica homologs, have enriched RNA processing function. In conclusion, O. viverrini and C. sinensis have 31 and 22 genes, respectively, that may be involved in their carcinogenic action through a direct targeting on the host cell nuclei., Opisthorchis viverrini; Clonorchis sinensis; Fasciola hepatica; In silico; Secretion; Nuclear targeting; Cancer.

Clonorchiasis is an important food-borne parasitic disease in China because of the popularity in ingesting raw freshwater fish. To explore the epidemiology and determinants of clonorchiasis in children, a cross-sectional survey was implemented in two middle schools in Qiyang county, Hunan province, in southeastern China. Questionnaire survey and fecal examination were implemented. Questionnaires were fed back by 627 students, while stool samples were collected from 557 students, out of which 545 ones also provided questionnaire information. The percentage of students ingesting raw freshwater fish was 40.5% (254/627), while the prevalence with Clonorchis sinensis infection was 18.9% (105/557). Such factors contributed significantly to the students' practice in eating raw freshwater fish including boys, fathers' eating raw freshwater fish, mothers' eating raw freshwater fish, and preparation of raw freshwater fish at home, with an adjusted odds ratio of 1.9 (95% confidence intervals (95% CI): 1.3-2.8), 3.9 (95% CI: 2.3-6.5), 3.0 (95% CI: 1.8-4.8) and 2.8 (95% CI: 1.8-4.5), correspondingly. Ingestion of raw freshwater fish, fathers' eating raw freshwater fish and preparation of raw freshwater fish at home were risk factors of C. sinensis infection in students, and the adjusted odds ratio was 3.2 (95% CI: 1.9-5.5), 2.1 (95% CI: 1.1-3.9) and 1.8 (95% CI: 1.0-3.2), respectively. Thus, clonorchiasis was endemic in the surveyed schools due to the ingestion of raw freshwater fish, which is influenced by family environment. Education should be implemented in schools to promote behavioral change of eating raw freshwater fish.

Neglected tropical diseases cause substantial morbidity and mortality in animals and people globally. Opisthorchiasis is one such disease, caused by the carcinogenic, Asian liver fluke, Opisthorchis viverrini. This hepatobiliary disease is known to be associated with malignant cancer (cholangiocarcinoma, CCA) and affects millions of people in Asia, including Thailand, Lao People's Democratic Republic (PDR) and Cambodia. No vaccine is available, and only one drug (praziquantel) is routinely employed against the parasite. Relatively little is known about the molecular biology of the fluke itself and the disease complex that it causes in humans. With the advent of high-throughput nucleic acid sequencing and bioinformatic technologies, it has now become possible to gain global insights into the molecular biology of parasites. The purpose of this minireview is (i) to discuss recent progress on the genomics of parasitic worms, with an emphasis on the draft genome and transcriptome of O. viverrini; (ii) to use results from an integrated, global analysis of the genomic and transcriptomic data, to explain how we believe that this carcinogenic fluke establishes in the biliary system, how it feeds, survives and protects itself in such a hostile, microaerobic environment within the liver, and to propose how this parasite evades or modulates host attack; and (iii) to indicate some of the challenges, and, more importantly, the exciting opportunities that the 'omic resources for O. viverrini now provide for a plethora of fundamental and applied research areas. Looking ahead, we hope that this genomic resource stimulates vibrant and productive collaborations within a consortium context, focused on the effective control of opisthorchiasis.

Autonomous retrotransposons, in which replication and transcription are coupled, encode the essential gag and pol genes as a fusion or separate overlapping form(s) that are expressed in single transcripts regulated by a common upstream promoter. The element-specific expression strategies have driven development of relevant translational recoding mechanisms including ribosomal frameshifting to satisfy the protein stoichiometry critical for the assembly of infectious virus-like particles. Retrotransposons with different recoding strategies exhibit a mosaic distribution pattern across the diverse families of reverse transcribing elements, even though their respective distributions are substantially skewed towards certain family groups. However, only a few investigations to date have focused on the emergence of retrotransposons evolving novel expression strategy and causal genetic drivers of the structural variants. In this study, the bulk of genomic and transcribed sequences of a Ty3/gypsy-like CsRn1 retrotransposon in Clonorchis sinensis were analyzed for the comprehensive examination of its expression strategy. Our results demonstrated that structural variants with single open reading frame (ORF) have recurrently emerged from precedential CsRn1 copies encoding overlapping gag-pol ORFs by a single-nucleotide insertion in an upstream region of gag stop codon. In the parasite genome, some of the newly evolved variants appeared to undergo proliferative burst as active master lineages together with their ancestral copies. The genetic event was similarly observed in Opisthorchis viverrini, the closest neighbor of C. sinensis, whereas the resulting structural variants might have failed to overcome purifying selection and comprised minor remnant copies in the Opisthorchis genome.

Clonorchis sinensis ( C. sinensis ) is a fish-borne trematode. Human can be infected by ingestion of C. sinensis metacercariae parasitized in grass carp ( Ctenopharyngodon idella ). For induction of effective oral immune responses, spores of Bacillus subtilis ( B. subtilis ) WB600 were utilized as vehicle to delivery Cs CP (cysteine protease of C. sinensis ) cooperated with CotC ( B.s -CotC-CP), one of coat proteins, to the gastrointestinal tract. After routine culture of 8–12 h in LB medium, B. subtilis containing CotC- Cs CP was transferred into the sporulation culture medium. SDS-PAGE, western blotting and the growth curve indicated that the best sporulation time of recombinant WB600 was 24–30 h at 37 °C with continuous shaking (250 rpm). Grass carp were fed with three levels of B.s -CotC-CP (1 × 10 6 , 1 × 10 7 , and 1 × 10 8 CFU g −1 ) incorporated in the basal pellets diet. The commercial pellets or supplemented with spores just expressing CotC (1 × 10 7 CFU g −1 ) were served as control diet. Our results showed that grass carp orally immunized with the feed-based B.s -CotC-CP developed a strong specific immune response with significantly ( P Cs CP were found in hindgut that is conducive to absorption and presentation of antigen. Moreover, B. subtilis spores appeared to show no sign of toxicity or damage in grass carp. Our cercariae challenge experiments suggested that oral administration of spores expressing Cs CP could develop an effective protection against C. sinensis in fish body. Therefore, this study demonstrated that the feed-based recombinant spores could trigger high levels of mucosal and humoral immunity, and would be a promising candidate vaccine against C. sinensis metacercariae formation in freshwater fish.

Clonorchis sinensis is major fish-borne trematode, endemic in North Vietnam. Risk factors described so far include individual eating behaviors and environmental factors. Here, additional to conventional risk factors, we report on socially influenced liver fluke transmission in endemic communities. A cross-sectional study on risk factors and fish sharing networks was conducted in 4 villages of Gia Thinh Commune, Ninh Binh Province. A total of 510 residents in 272 households were recruited for risk factor analysis while 220 households, 28 fishermen and 10 fish-sellers were enrolled for social network study. Fecal examination for C. sinensis eggs was performed. Average C. sinensis infection rate at Gia Thinh commune was 16.5% (range 2% to 34.4%). Higher infection rates were significantly associated with males, lower educational levels, eating raw fish, and location of the villages. Social network analysis (SNA) showed a strong positive correlation between ego network size (number of households in fish sharing network) and quantity of raw fish consumed (r=0.603, P< 0.05). The infection rate in people who ate raw-fish caught from a nearby river was significantly higher than those who consumed fish taken from farmed ponds (P

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory–secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory–secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory–secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory–secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory–secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory–secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma.

Clonorchiasis, caused by the consumption of raw or undercooked freshwater fish containing infective metacercariae of Clonorchis sinensisis (C.sinensis), remains a common public health problem. New effective prevention strategies are still urgent to control this food-borne infectious disease. The previous studies suggested Bacillus subtilis (B. subtilis) spores was an ideal vaccines delivery system, and the C.sinensis enolase (CsENO) was a potential vaccine candidate against clonorchiasis. In the current study, we detected CsENO-specific IgM levels by ELISA in sera, intestinal mucus and skin mucus in grass carps (Ctenopharyngodon idella) through oral administration with B. subtilis spores surface expressing CsENO. In addition, immune-related genes expression was also measured by qRT-PCR. Grass carps orally treated with B. subtilis spores or normal forages were used as controls. The results of ELISA manifested that specific IgM levels of grass carps in CsENO group in sera, intestine mucus and skin mucus almost significantly increased from week 4 post the first oral administration when compared to the two control groups. The levels of specific IgM reached its peak in intestine mucus firstly, then in sera, and last in skin mucus. qRT-PCR results showed that 5 immune-related genes expression had different degree of rising trend in CsENO group when compared to the two control groups. Our study demonstrated that orally administrated with B. subtilis spores expressing CsENO induced innate and adaptive immunity, systemic and local mucosal immunity, and humoral and cellular immunity. Our work may pave the way to clarify the exact mechanisms of protective efficacy elicited by B. subtilis spores expressing CsENO and provide new ideas for vaccine development against C. sinensis infection.

To effectively promote the implementation of interventions, the identification of high-risk groups and the characteristics of Clonorchis sinensis infection in endemic regions are needed. In a clonorchiasis-endemic area, local residents were randomly enrolled for helminth egg examination in June 2016. The prevalence in subpopulations as well as the knowledge, attitudes, and behaviours and the factors influencing clonorchiasis in egg-positive populations were analysed. A total of 2282 local residents participated in the survey; the C. sinensis prevalence was 48.6% (1109 persons). A higher prevalence was found in males (62.6%) than in females (29.7%). People older than 30 years had the highest prevalence (52.7%–57.6%). Among the 888 persons who were infected with C. sinensis and participated the questionnaire investigation, 19.0% (169/888) knew that it could cause cancer. In addition, 60.6% of people reported that they intended to keep eating raw fish despite knowing the risk of infection. The two primary reasons for continuing to eat raw fish were the disease being regarded as not serious (38.3%) and the belief that anti-parasite medications are effective (39.6%). A total of 94.4% (797/844) of responders reported eating raw fish more frequently in the home than outside of the home. Our study revealed a notably high C. sinensis prevalence in the study area. Awareness of clonorchiasis disease severity should be increased among high-risk individuals and families in highly endemic areas., Highlights • The overall prevalence of Clonorchis sinensis was high in our study area. • Males and people over 30 were more likely infected by Clonorchis sinensis. • Limited proportion of infected people knew that Clonorchis sinensis could lead to cancer. • People tended to keep eating raw fish even knowing it caused clonorchiasis.

The surface tegument of Fasciola hepatica is a crucial tissue due to its key role at the host-parasite interface. We characterised three novel proteins, termed Fhteg1, Fhteg5 and Fhteg8, that are found in the tegument membrane fraction of adult F. hepatica. Bioinformatic analysis of proteomic datasets identified Fhteg5 and Fhteg8 as tegumental glycoproteins and revealed that Fhteg1, Fhteg5 and Fhteg8 are associated with exosomes of adult F. hepatica. Fhteg1, Fhteg5 and Fhteg8 appear to be related to uncharacterised sequences in F. gigantica, Fasciolopsis buski, Echinostoma caproni, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma japonicum and S. mansoni, although F. hepatica appears to have expanded this family. Fhteg1 and Fhteg5 were characterised in detail. The Fhteg1 and Fhteg5 gene transcripts each demonstrate significant upregulation in juvenile fluke 2-4 days post-excystment, with transcript levels maintained during development over 3 weeks in vitro. RNAseq data showed that both Fhtegs are expressed in the adult life stage, although the transcript levels were about 8 fold lower than those in juveniles (3 week post infection). Using immunocytochemistry, Fhteg1 and Fhteg5 were each shown to be expressed in cells adjacent to the muscle layer as well as on the surface of 1 week old juveniles, whilst Fhteg5 was also present in cells at the base of the pharynx. RNAi mediated knockdown of Fhteg1 and Fhteg5 transcripts in 4-10 day old juveniles had no effect on parasite survival, movement or growth in vitro. Although no IgG responses were observed for Fhteg1 or Fhteg5 during infection in sheep and cattle, both proteins elicited a low IgG response in a proportion of infected rats. Rats vaccinated with Fhteg1 and Fhteg5 showed good IgG responses to both proteins and a mean 48.2 % reduction in worm burden following parasite challenge. Although vaccination of cattle with both proteins induced a range of IgG responses, no protection was observed against parasite challenge. This is the first study to provide insights into the molecular properties of two novel, developmentally regulated surface tegument proteins in F. hepatica.

Stimulated largely by the availability of new technology, biomedical research at the molecular-level and chemical-based control approaches arguably dominate the field of infectious diseases. Along with this, the proximate view of disease etiology predominates to the exclusion of the ultimate, evolutionary biology-based, causation perspective. Yet, historically and up to today, research in evolutionary biology has provided much of the foundation for understanding the mechanisms underlying disease transmission dynamics, virulence, and the design of effective integrated control strategies. Here we review the state of knowledge regarding the biology of Asian liver Fluke-host relationship, parasitology, phylodynamics, drug-based interventions and liver Fluke-related cancer etiology from an evolutionary biology perspective. We consider how evolutionary principles, mechanisms and research methods could help refine our understanding of clinical disease associated with infection by Liver Flukes as well as their transmission dynamics. We identify a series of questions for an evolutionary biology research agenda for the liver Fluke that should contribute to an increased understanding of liver Fluke-associated diseases. Finally, we describe an integrative evolutionary medicine approach to liver Fluke prevention and control highlighting the need to better contextualize interventions within a broader human health and sustainable development framework.

Clonorchis sinensis (C. sinensis) infection can lead to biliary fibrosis. MicroRNAs (miRNAs) play important roles in regulation of genes expression in the liver diseases. However, the differential expression of miRNAs that probably regulates the portal fibrogenesis caused by C. sinensis has not yet been investigated. Hepatic miRNAs expression profiles from C. sinensis-infected mice at different time-points were analyzed by miRNA microarray and validated by quantitative real-time PCR (qRT-PCR). 349 miRNAs were differentially expressed in the liver of the C. sinensis-infected mice at 2, 8 or 16weeks post infection (p.i.), compared with those at 0week p.i., and there were 143 down-regulated and 206 up-regulated miRNAs among them. These all dysregulated miRNAs were potentially involved in the pathological processes of clonorchiasis by regulation of cancer-related signaling pathway, TGF-β signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, PI3K /AKT signaling pathway, etc. 169 of these dysregulated miRNAs were predicted to be involved in the TGF/Smads signaling pathway which plays an important role in the biliary fibrosis caused by C. sinensis. Additionally, miRNA-32, miRNA-34a, miRNA-125b and miRNA-497 were negatively correlated with Smad7 expression, indicating these miRNAs may specifically down-regulate Smad7 expression and participate in regulation of biliary fibrosis caused by C. sinensis. The results of the present study for the first time demonstrated that miRNAs were differentially expressed in the liver of mice infected by C. sinensis, and these miRNAs may play important roles in regulation of peribiliary fibrosis caused by C. sinensis, which may provide possible therapeutic targets for clonorchiasis.

Clonorchis sinensis is a high-risk pathogenic helminth that strongly provokes inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma in chronically infected individuals. Chronic inflammation is associated with an increased risk of various cancers due to the disruption of redox homeostasis. Accordingly, the present study was conducted to examine the time course relationship between histopathological changes and the appearance of oxidative stress markers, including lipid peroxidation, enzymes involved in lipid peroxidation, and mutagenic DNA adducts in the livers of mice infected with C. sinensis, as well as proinflammatory cytokines in infected mouse sera. Histopathological phenotypes such as bile duct epithelial hyperplasia, periductal fibrosis, edema and inflammatory infiltration increased in infected livers in a time-dependent manner. Intense immunoreactivity of lipid peroxidation products (4-hydroxy-2-nonenal; malondialdehyde), cyclooxygenase-2, 5-lipoxygenase and 8-oxo-7,8-dihydro-2'-deoxyguanosine were concomitantly observed in these injured regions. We also found elevated expressions of cyclooxygenase-2 and 5-lipoxygenase in C. sinensis excretory-secretory product-treated cholangiocarcinoma cells. Moreover, the levels of proinflammatory cytokines such as TNF-α, ILβ-1 and IL-6 were differentially upregulated in infected sera. With regard to oxidative stress-mediated carcinogenesis, our findings suggest that C. sinensis infestation may disrupt host redox homeostasis, creating a damaging environment that favors the development of advanced hepatobiliary diseases such as clonorchiasis-associated cholangiocarcinoma.

Clonorchis sinensis infection is caused by ingestion of raw freshwater fish containing the infective larvae of Clonorchis sinensis. It is highly endemic in East Asia, especially in China. Selective chemotherapy of people who report habitual eating of raw freshwater fish is a control measure. As the performance of this screening technique has not yet been fully evaluated in China, a cross-sectional study was conducted, covering 17 counties in four major clonorchiasis-endemic provinces. About 1 000 participants were enrolled from each county. Fecal samples were collected and examined for helminth eggs and each person enrolled was asked about their practice with respect to eating raw freshwater fish. In total, 16 230 participants from 16 counties were finally included. The overall prevalence of C. sinensis infection was 10.8%, ranging from 0 to 53.7% in the 16 counties, while the percentage of inhabitants eating raw freshwater fish was 26.5%, ranging from 0 to 79.1%. The overall sensitivity and specificity of screening for C. sinensis infection in this approach was 82.3% and 80.3%, respectively, yielding a Youden's index of 0.6. The overall positive and negative likelihood ratios were 4.2 and 0.2, respectively, while the overall positive and negative predictive values were 33.5% and 97.4%, respectively. Furthermore, the sensitivity was higher with regard to high-intensity infections compared to light infections.

Clonorchis sinensis is a fish-borne trematode that inhabits the bile duct of mammals including humans. Clonorchiasis is prevalent in China, Korea, and Vietnam, and 15-20 million people are estimated to be infected by this fluke. Freshwater snails act as the first intermediate host for the proliferation of C. sinensis larvae and shed the cercariae into water. The cercariae penetrate the skin of freshwater fish and transform to metacercariae. Humans are infected by eating raw or undercooked freshwater fish as dishes of filet, "sashimi," or congee, which contain C. sinensis metacercariae. In humans, the C. sinensis metacercariae excyst in the duodenum, and juvenile flukes migrate up via bile chemotaxis into bile ducts. Once there, C. sinensis provokes hyperplasia of the bile duct epithelium, obstructive jaundice, ascites, liver enlargement and cirrhosis, and infrequent cholangiocarcinoma (CCA). Although the association between C. sinensis infection and CCA has been firmly established in past decades, the underlying mechanisms are not elucidated in detail. In the context of chronic clonorchiasis-associated hepatobiliary aberrations, the constitutive disruption of redox homeostasis and dysregulation of physiological signaling pathways may promote the malignant transformation of cholangiocytes, thus leading to substantial acquisition of a more aggressive phenotype by these cells: CCA. With advances of genomic and molecular biological approaches, diverse C. sinensis proteins that are essential for parasite physiology and pathogenicity have been identified and characterized. Some of the proteins have been considered as attractive targets for development of vaccines and chemotherapeutics. Candidate antigens for reliable serodiagnosis of clonorchiasis have been studied.

China was once a country plagued by parasitic diseases. At the beginning of the founding of the People's Republic of China, nearly 80% of the population suffered from parasitic diseases because of poverty and poor sanitary conditions. After nearly 70 years of development, China has made remarkable achievements in the prevention and control of parasitic diseases, and the prevalence of parasitic diseases has been greatly reduced. In addition to organizational leadership from the government and various preventive measures, drug treatment and drug research & development are important and irreplaceable links in prevention and control work. Since the 1950s, China has begun to introduce, produce and imitate antiparasitic drugs from abroad, such as santonin, benzimidazole, and praziquantel. Chinese scientists have also contributed to the optimization of production techniques, improvements in drug formulation, the application in the clinic and the mechanisms of actions of generic drugs. At the same time, China has independently developed tribendimidine (TrBD, a broad spectrum anthelminthic), and its anthelminthic spectrum has been comprehensively studied. It is active against almost 20 parasites, is especially superior to benzimidazoles against Necator americanus, and surpasses the effectiveness of praziquantel against Clonorchis sinensis. In the treatment of tapeworm disease, the traditional Chinese medicines pumpkin seeds and betel nuts have good curative effects for taeniasis. Chinese scientists have explored the action modes and clinical administration methods of pumpkin seeds and betel nuts, which is still the main clinical regimen for the disease. This paper reviews the history and progress of the study of anthelmintics in intestinal helminth infections since the founding of the People's Republic of China and aiming to support clinicians and drug researchers in China and other countries.

Fasciolosis, a foodborne zoonotic disease, caused by Fasciola species which is considered an important problem for human health and livestock husbandry development. Snails are intermediate hosts of F. hepatica, the epidemiological surveillance of snails can evaluate the transmission risk of this disease in human and livestock. In this study, we developed a nest-polymerase chain reaction (nest-PCR) to detect the DNA of F. hepatica in Radix cucunorica, a prevalent intermediate host of this parasite in northwestern China. The nest-PCR was used to amplify a 208 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica with two pairs of primers. The method was able to detect up to 0.16 fg genomic DNA in a 25 μL PCR reaction system even effected with high concentrations of snail DNA, and no cross reaction was observed from the genomic DNA of Paramphistomum cervi, Clonorchis sinensis, Orientobilharzia turkestanicum, Metorchis orientalis, Dicrocoelium chinensis. To evaluate the transmission risk of this disease, 409 snail samples collected from different areas of Gansu province were used to detect and analyze the transmission risk of F. hepatica in this area. Of 409 snail samples, the overall prevalence is 43.76%. The prevalence was 92.75% in Gannan Tibetan Autonomous Prefecture, while no snail was positive for F. hepatica in Linxia Hui Autonomous Prefecture. The nest-PCR was firstly used to detect the infection of F. hepatica in snail. It is a novel, useful and convenient method with high sensitivity and specificity. This study is the first report about the epidemiological surveillance of snail infection by F. hepatica in northwestern China, which will help to evaluate the transmission risk of F. hepatica in northwestern China.

Asparaginyl endopeptidases (AEP: EC 3.4.22.34) are a family of cysteine proteases classified into the MEROPS clan CD, family C13. In this study, we characterized the biochemical and antigenic properties of an AEP of Clonorchis sinensis (CsAEP). The recombinant CsAEP showed hydrolytic activity at pH values ranging from acidic to neutral with optimum activity at pH 6.0. While the recombinant CsAEP was stable at neutral pHs, it was unstable at acidic pHs and resulted in loss of enzymatic activity. The recombinant enzyme was effectively inhibited by iodoacetic acid and N-ethylmaleimide, but not by E-64. The partially purified native CsAEP showed biochemical properties similar to the recombinant enzyme. Native CsAEP is likely to be cleaved into an N-terminal mature enzyme and a C-terminal fragment via autocatalytic activation at acidic pHs. Polyclonal antibody raised against the recombinant CsAEP recognized three forms of CsAEP, proenzyme, the N-terminal mature enzyme and the C-terminal fragment, in the worm extract (WE) of C. sinensis. However, only the C-terminal fragment was mainly found in the excretory and secretory (ES) products of the parasite. Strong CsAEP activity was found in the WE, but only a trace level of CsAEP activity was detected in the ES products of the parasite. CsAEP was expressed in various developmental stages of C. sinensis, from metacercariae to adults, and was found to be localized in the intestine of the parasite as well as in intestinal contents. Sera from rats experimentally infected with C. sinensis reacted with CsAEP beginning 4 weeks after infection. These results suggest that CsAEP is a gut-associated enzyme synthesized in the intestine of C. sinensis and subsequently secreted into the intestinal lumen of the parasite.

Paramyosin of Clonorchis sinensis (CsPmy) is a myofibrillar protein localized in subtegumental muscle, tegument, and the muscle layer surrounding the intestine of the parasite. Previously, we have identified that CsPmy reacted with sera of human clonorchiasis and this protein had a potential as a candidate antigen for serodiagnosis of clonorchiasis. However, we also found that CsPmy is able to bind to human immunoglobulin G (IgG) in non-specific manners, which can affect the diagnostic value of the protein. Here, we mapped CsPmy-specific IgG binding site on CsPmy to analyze the putative epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis. The fragmental expression of CsPmy followed by immunoblot analyses with sera from patients with clonorchiasis and non-specific human IgG revealed that the middle portion of CsPmy (CsPmyC: 301-600 amino acid residues) had epitopes responsible for CsPmy-specific IgG recognition. The precise CsPmy-specific IgG binding site was further narrowed down to a fragment (CsPmyC-2), which harbors 151 amino acid residues (375-525) of CsPmy. Specific antibodies for CsPmyC-2 were produced in rats after two-weeks of post-experimental infection. The CsPmyC-2 showed low levels of cross reactivity against the sera from patients with other helminth parasites. Our results suggested that CsPmyC-2 has real epitopes recognized by CsPmy-specific IgG in sera of human clonorchiasis and the fragment can be useful as a reliable serodiagnostic antigen to develop a serodiagnostic method for clonorchiasis.

Objective: To characterize immunogenic protein fraction of Clonorchis sinensis (C. sinensis) by partial purification. Methods: A total of 30 hamsters were infected with 50 C. sinensis metacercariae, and then C. sinensis protein was purified by gel filtration chromatography. Indirect ELISA and immunoblot were used to detect the antibody in sera of hamsters infected with C. sinensis. Results: The gel filtration showed 2 peaks at high (fraction No. 10 to 14) and low (fraction No. 21 to 26) molecular weight proteins. Indirect ELISA showed that both antibodies of clonorchiasis and opisthorchiasis reacted strongly with early fractions (6 to 14) and the reaction was gradually reduced at middle and late fractions (15 to 50). Both antibodies showed different individual fraction of C. sinensis by immunoblot. It showed several protein bands that the 34 and 37 kDa were major proteins. The 53 kDa protein which was only found in the clonorchiasis reacted with fraction 20. Conclusions: The purified antigen of C. sinensis reacted similarly with both antibodies of clonorchiasis and opisthorchiasis where strong reaction was seen with early fractions. The C. sinensis protein fraction No. 20 may be useful for immunodiagnosis of clonorchiasis.

Clonorchiasis is a parasitic disease of high public health importance in many countries in southeastern Asia and is caused by the Chinese liver fluke Clonorchis sinensis. However, the genetic structure and demographic history of its populations has not been sufficiently studied throughout the geographic range of the species and available data are based mainly on partial gene sequencing. In this study, we explored the genetic diversity of the complete 1560 bp cytochrome c oxidase subunit 1 (cox1) gene sequence for geographically isolated C. sinensis populations in Russia and Vietnam, to our knowledge for the first time. The results demonstrated low nucleotide and high haplotype differentiation within and between the two compared regions and a clear geographical vector for the distribution of genetic diversity patterns among the studied populations. These results suggest a deep local adaptation of the parasite to its environment including intermediate hosts and the existence of gene flow across the species' range. Additionally, we have predicted an amino acid substitution in the functional site of the COX1 protein among the Vietnamese populations, which were reported to be difficult to treat with praziquantel. The haplotype networks consisted of several region-specific phylogenetic lineages, the formation of which could have occurred during the most extensive penultimate glaciations in the Pleistocene Epoch. The patterns of genetic diversity and demographics are consistent with population growth of the liver fluke in the late Pleistocene following the Last Glacial Maximum, indicating the lack of a population bottleneck during the recent past in the species' history. The data obtained have important implications for understanding the phylogeography of C. sinensis, its host-parasite interactions, the ability of this parasite to evolve drug resistance, and the epidemiology of clonorchiasis under global climate change.

The present study focussed on investigating CD59-like molecules of Fasciola hepatica. A cDNA encoding a CD59-like protein (termed FhCD59-1) identified previously in the membrane fraction of the F. hepatica tegument was isolated. This homologue was shown to encode a predicted open reading frame (ORF) of 122 amino acids (aa) orthologous to human CD59 with a 25 aa signal peptide, a mature protein containing 10 cysteines and a conserved CD59/Ly-6 family motif "CCXXXXCN". An analysis of cDNAs from two different adult specimens of F. hepatica revealed seven variable types of FhCD59-1 sequences, designated FhCD59-1.1 to FhCD59-1.7, which had 94.3-99.7% amino acid sequence identity upon pairwise comparison. Molecular modeling of FhCD59-1.1 with human CD59 confirmed the presence of the three-finger protein domain found in the CD59 family and predicted three disulphide bonds in the F. hepatica sequence. The interrogation of F. hepatica databases identified two additional sequences, designated FhCD59-2 and FhCD59-3, which had only 23.4-29.5% amino acid identity to FhCD59-1.1. Orthologues of the inferred CD59 protein sequences of F. hepatica were also identified in other flatworms, including Fasciola gigantica, Fascioloides magna, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Clonorchis sinensis, Opisthorchis viverrini, Taenia solium, Echinococcus granulosus and the free living Schmidtea mediterannea. The results revealed a considerable degree of sequence complexity in the CD59-like sequence families in F. hepatica and flatworms. Phylogenetic analysis of CD59-like aa sequences from F. hepatica and flatworms showed that FhCD59-2 clustered with the known surface-associated protein SmCD59-2 of S. mansoni. Relatively well-supported clades specific to schistosomes, fasciolids and opisthorchiids were identified. The qPCR analysis of gene transcription showed that the relative expression of these 3 FhCD59-like sequences varied by 11-47-fold during fluke maturation, from the newly excysted juvenile (NEJ) to the adult stage. These findings suggest that different FhCD59-like sequences play distinct roles during the development of F. hepatica.

The prevalence of Clonorchis sinensis infection in freshwater fishes was surveyed in Heilongjiang Province, northeastern China, between August 2011 and September 2013. Thirteen species of freshwater fish (n=3221) and one species of shrimp (n=93) were collected from Songhua river, Nenjiang river and other lakes or ponds in 37 sites of 15 representative cities in Heilongjiang Province. They were individually examined by digestion technique, and the C. sinensis metacercariae were identified morphologically followed by confirmation using sequences of the second internal transcribed spacer of ribosomal DNA. Ten of the 13 examined species of freshwater fishes were infected with C. sinensis metacercariae, while all shrimps were negative. The overall prevalence of C. sinensis infection in 3221 examined freshwater fishes was 19.96%, with 42.57% (272/639) in Pseudorasbora parva, 22.55% (83/368) in Hemicculter leuciclus, 20.44% (121/592) in Carassius auratus, 17.71% (68/384) in Saurogobio dabryi, 10.85% (23/212) in Rhodeus ocellatus, 10.54% (48/455) in Phoxinus lagowskii, 8.20% (21/256) in Perccottus glehnii, 6.25% (5/80) in Misgurnus anguillicaudatus, 4.55% (1/22) in Xenocypris davidi, and 1.49% (1/67) in Cyprinus carpio. The average infection intensity in P. parva was 103.3 encysted metacercariae per gram of fish meat in Zhaoyuan city. The average prevalence of C. sinensis infection in Songhua river, Nenjiang river and lakes or ponds were 31.96% (503/1574), 11.30% (102/903) and 7.93% (59/744), respectively. The prevalence of C. sinensis infection in Zhaoyuan city (43.68%) was the highest among all sampling locations. These results revealed a high-prevalence of C. sinensis infection in freshwater fishes in Heilongjiang Province, northeastern China, posing significant public health concern.

Previous studies demonstrated that Clonochis sinensis-derived crude antigens suppress development of allergic responses. We investigated the effects of C. sinensis venom allergen-like (CsVAL) proteins on immune-modulating activities in allergic inflammatory response. Using RBL-2H3 rat mast cells, we demonstrated that CsVAL inhibits antigen-induced β-hexosaminidase release from immunoglobulin E-sensitized RBL-2H3 cells, and this inhibitory activity occurs by suppressing Lyn phosphorylation and intracellular reactive oxygen species production. In addition, CsVAL peptide treatment inhibits activation of protein kinase C-α and extracellular signal-regulated kinase 1/2, which are involved in degranulation of immunoglobulin E-sensitized mast cells. Furthermore, immunization with CsVAL suppressed development of skin inflammation by assessing ear thickness and cutaneous infiltration by eosinophils and mast cells in oxazolone-induced contact hypersensitivity in vivo mouse model. These results suggest that CsVAL is a promising candidate as an effective mast cell inhibitor for allergic and inflammatory diseases.

Clonorchis sinensis (C. sinensis) infections remain the common public health problem in freshwater fish consumption areas. New effective prevention strategies are still the urgent challenges to control this kind of foodborne infectious disease. The biochemical importance and biological relevance render C. sinensis enolase (Csenolase) as a potential vaccine candidate. In the present study, we constructed Escherichia coli/Bacillus subtilis shuttle genetic engineering system and investigated the potential of Csenolase as an oral vaccine candidate for C. sinensis prevention in different immunization routes. Our results showed that, compared with control groups, both recombinant Csenolase protein and nucleic acid could induce a mixed IgG1/IgG2a immune response when administrated subcutaneously (P

Tyrosinase (TYR) is a copper-containing glycoenzyme that mediates hydroxylation of tyrosine into dihydroxyphenylalanine and oxidation of dihydroxyphenylalanine into dihydroxyphenylalanine quinone. TYRs play pivotal roles in eggshell sclerotisation of trematode parasites, while their comprehensive biochemical properties remain elusive. We characterised genes encoding four TYRs (CsTYR1–4) of Clonorchis sinensis, a causative agent of human hepatobiliary disease. These genes shared tightly conserved amino acid residues, two copper binding catalytic motifs and a cysteine-rich epidermal growth factor-like domain. The native and recombinant CsTYRs showed high reactivity against diphenol compounds, especially those with hydroxyl groups in ortho-positions (catechol and l -dihydroxyphenylalanine), but showed minimal activity toward monophenol compounds. Diphenolase activity was enhanced by increased pH of the reaction buffer from 5.0 to 7.0. The temporal induction of CsTYR expression coordinated with the sexual maturation of the worm; enzyme activity was mainly in the vitelline glands and intrauterine immature eggs proximal to the ovary. The primary structures and functional domains of CsTYRs showed significant similarities to those of the vertebrate orthologs, whereas the amino acids shared with the nematode and insect proteins were largely restricted in the bicopper active center. Unlike highly diverged TYR homologs in vertebrates, multiple paralogs have not yet evolved into the separate lineages in trematode genomes, suggesting that duplication of TYR genes might relate to increased genic dosage/redundancy in trematodes. In vitro treatment of copper chelator, diethyldithiocarbamic acid, inhibited generation of phenotypically normal egg. TYR proteins are essential for C. sinensis reproduction, thus might be targeted for therapeutic and vaccine strategies against clonorchiasis, which is prevalent in several Asian countries and is one of the most important predisposing factors for human cholangiocarcinoma. The close phylogenetic relationships between trematode and vertebrate homologs also provide a molecular clue to understand the multifaceted evolutionary pathway of TYR homologs across animal taxa.

The crystal structures of CsGST in two different space groups revealed that Asp26 and His79 coordinate a zinc ion. In one space group, His46 of an adjacent molecule participates in the coordination within 2.0Å. In the other space group, Asp26, His79 and a water molecule coordinate a zinc ion. The CsGST-D26H structure showed that four histidine residues - His26 and His79 from one molecule and the same residues from a symmetry-related neighboring molecule - coordinate a zinc ion. The coordinated zinc ions are located between two molecules and mediate molecular contacts within the crystal.

CsCF-6 is a member of the multigene family of cathepsin F cysteine proteases of Clonorchis sinensis . Similar to other papain family proteases, CsCF-6 is synthesized as a proenzyme and is converted to the mature form by autocatalytic removal of the prodomain. Here, we analyzed the regulatory and inhibitory elements within the CsCF-6 prodomain to understand the regulatory mechanism of CsCF-6 by its prodomain. The CsCF-6 prodomain played an essential role in the folding of CsCF-6. Particularly, the ERFNAQ motif within the prodomain was essential, and the minimum segment required for this event was the C-terminal part of the prodomain, including Asn 58 and downstream residues. The CsCF-6 prodomain effectively inhibited CsCF-6, in which the ERFNAQ motif played a critical role in the inhibition, but the GTFD motif was also required for complete inhibition of CsCF-6. The CsCF-6 prodomain showed broad inhibitory activity against several cysteine proteases. These results suggest that the CsCF-6 prodomain plays bi-functional roles in correct folding and inhibition of its cognate enzyme.

We describe here a fatal case of a dog with extensive Clonorchis sinensis (C. sinensis) infection. C. sinensis were detected in organs including the abdomen, bladder and heart. The infection was very heavy with a total number of 155,183 worms. These worms were in different developmental stages, but the majority of them were adult.

In the present study, Clonorchis sinensis ( C. sinensis ) ancient DNA (aDNA) was successfully extracted from human remains discovered in a tomb dating to the medieval Joseon dynasty of Korea. The presence of C. sinensis eggs was confirmed by microscopic observation, after which a PCR-based aDNA analysis was performed using primer sets designed for the amplification of nuclear ribosomal internal transcribed spacer 1 (ITS1), 2 (ITS2) and mitochondrial cytochrome c oxidase 1 (CO1) genes. The sequences obtained were 100% homologous to some contemporary C. sinensis gene sequences reported from Korea and other East Asian countries. We believe that the results of our analysis expand the temporal and geographical scopes of research on the history of C. sinensis infection in different human populations.

The present study examined the molecular organisation and sequence variation in the nuclear ribosomal DNA (rDNA) region, including the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the Clonorchis sinensis from the Russian Far East. The relevant sequences from other parts of this species' area were downloaded from GenBank. The results showed 100% identity for all investigated 5.8S-ITS2 rDNA sequences. In contrast, two levels of intraspecific variations were revealed in the complete ITS1 sequences. The intra-genomic variation resulted from a C/T polymorphism in a single position. The inter-individual differences between the ITS1 sequences were both due to nucleotide and size polymorphisms resulting from a varying number of five-nucleotide repeats and followed by two ITS1 length variants. These variant frequencies correlate with the clonorchiasis level in some geographical localities. ITS1 differences, both in the mutation profile and mutation localisation, were revealed between northern and southern geographical samples. The presence of GC boxes that are identical to known regulatory motifs in eukaryotes was detected within the ITS1 sub-repeats. The predicted secondary structures for ITS1 consist of two large branches, one of which was invariable, while another depended on ITS1 length. The predicted secondary structure for ITS2 includes four helices around the core. The main differences between C. sinensis and other opisthorchids were localised on the tops of helices 2, 3, and 4. A phylogenetic MST reconstruction subdivided all ITS1 sequences into two well differentiated clusters, each with the major widespread ribotype, and showed that ribotype diversity in both Russia and Korea is much lower than in China. The results obtained demonstrate the feasibility of complete ITS1 sequences in C. sinensis population genetics and can be considered as a basis for further studies of the parasite infection because they may help to elucidate the molecular mechanisms of pathogen evolution and adaptation.

An 18.2 kDa protein from the liver fluke, Fasciola hepatica has been identified and characterised. The protein shows strongest sequence similarity to egg antigen proteins from Schistosoma mansoni, Schistosoma japonicum and Clonorchis sinensis. The protein is predicted to adopt a calmodulin-like fold; it thus represents the third calmodulin-like protein to be characterised in F. hepatica and has been named FhCaM3. Compared to the classical calmodulin structure there are some variations. Most noticeably, the central, linker helix is disrupted by a cysteine residue. Alkaline native gel electrophoresis showed that FhCaM3 binds calcium ions. This binding event increases the ability of the protein to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, consistent with an increase in surface hydrophobicity as seen in other calmodulins. FhCaM3 binds to the calmodulin antagonists trifluoperazine and W7, but not to the myosin regulatory light chain binding compound praziquantel. Immunolocalisation demonstrated that the protein is found in eggs and vitelline cells. Given the critical role of calcium ions in egg formation and hatching this suggests that FhCaM3 may play a role in calcium signalling in these processes. Consequently the antagonism of FhCaM3 may, potentially, offer a method for inhibiting egg production and thus reducing the spread of infection.