Schistosomiasis, a parasite infectious disease caused by Schistosoma japonicum, often leads to egg granuloma and fibrosis due to the inflammatory reaction triggered by egg antigens released in the host liver. This study focuses on the role of the egg antigens CP1412 protein of S. japonicum (SjCP1412) with RNase activity in promoting liver fibrosis. In this study, the recombinant egg ribonuclease SjCP1412, which had RNase activity, was successfully prepared. By analysing the serum of the population, it has been proven that the anti-SjCP1412 IgG in the serum of patients with advanced schistosomiasis was moderately correlated with liver fibrosis, and SjCP1412 may be an important antigen associated with liver fibrosis in schistosomiasis. In vitro, the rSjCP1412 protein induced the human liver cancer cell line Hep G2 and liver sinusoidal endothelial cells apoptosis and necrosis and the release of proinflammatory damage-associated molecular patterns (DAMPs). In mice infected with schistosomes, rSjCP1412 immunization or antibody neutralization of SjCP1412 activity significantly reduced cell apoptosis and necroptosis in liver tissue, thereby reducing inflammation and liver fibrosis. In summary, the SjCP1412 protein plays a crucial role in promoting liver fibrosis during schistosomiasis through mediating the liver cells apoptosis and necroptosis to release DAMPs inducing an inflammatory reaction. Blocking SjCP1412 activity could inhibit its proapoptotic and necrotic effects and alleviate hepatic fibrosis. These findings suggest that SjCP1412 may be served as a promising drug target for managing liver fibrosis in schistosomiasis japonica.

Schistosomiasis has been subjected to extensive control efforts in the People's Republic of China (China) which aims to eliminate the disease by 2030. We describe baseline results of a longitudinal cohort study undertaken in the Dongting and Poyang lakes areas of central China designed to determine the prevalence of Schistosoma japonicum in humans, animals (goats and bovines) and Oncomelania snails utilizing molecular diagnostics procedures. Data from the Chinese National Schistosomiasis Control Programme (CNSCP) were compared with the molecular results obtained.

Sixteen villages from Hunan and Jiangxi provinces were surveyed; animals were only found in Hunan. The prevalence of schistosomiasis in humans was 1.8% in Jiangxi and 8.0% in Hunan determined by real-time polymerase chain reaction (PCR), while 18.3% of animals were positive by digital droplet PCR. The CNSCP data indicated that all villages harboured S. japonicum-infected individuals, detected serologically by indirect haemagglutination assay (IHA), but very few, if any, of these were subsequently positive by Kato-Katz (KK).

Based on the outcome of the IHA and KK results, the CNSCP incorporates targeted human praziquantel chemotherapy but this approach can miss some infections as evidenced by the results reported here. Sensitive molecular diagnostics can play a key role in the elimination of schistosomiasis in China and inform control measures allowing for a more systematic approach to treatment.

Novel tools for early diagnosis and monitoring of schistosomiasis are urgently needed. This study aimed to validate parasite-derived miRNAs as potential novel biomarkers for the detection of human Schistosoma japonicum infection. A total of 21 miRNAs were initially validated by real-time-polymerase chain reaction (RT-PCR) using serum samples of S. japonicum-infected BALB/c mice. Of these, 6 miRNAs were further validated with a human cohort of individuals from a schistosomiasis-endemic area of the Philippines. RT-PCR analysis showed that two parasite-derived miRNAs (sja-miR-2b-5p and sja-miR-2c-5p) could detect infected individuals with low infection intensity with moderate sensitivity/specificity values of 66%/68% and 55%/80%, respectively. Analysis of the combined data for the two parasite miRNAs revealed a specificity of 77.4% and a sensitivity of 60.0% with an area under the curve (AUC) value of 0.6906 (P = 0.0069); however, a duplex RT-PCR targeting both sja-miR-2b-5p and sja-miR-2c-5p did not result in an increased diagnostic performance compared with the singleplex assays. Furthermore, the serum level of sja-miR-2c-5p correlated significantly with faecal egg counts, whereas the other five miRNAs did not. Targeting S. japonicum-derived miRNAs in serum resulted in a moderate diagnostic performance when applied to a low schistosome infection intensity setting.

Schistosomiasis in China has been substantially reduced due to an effective control programme employing various measures including bovine and human chemotherapy, and the removal of bovines from endemic areas. To fulfil elimination targets, it will be necessary to identify other possible reservoir hosts for Schistosoma japonicum and include them in future control efforts. This study determined the infection prevalence of S. japonicum in rodents (0–9·21%), dogs (0–18·37%) and goats (6·9–46·4%) from the Dongting Lake area of Hunan province, using a combination of traditional coproparasitological techniques (miracidial hatching technique and Kato-Katz thick smear technique) and molecular methods [quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR)]. We found a much higher prevalence in goats than previously recorded in this setting. Cattle and water buffalo were also examined using the same procedures and all were found to be infected, emphasising the occurrence of active transmission. qPCR and ddPCR were much more sensitive than the coproparasitological procedures with both KK and MHT considerably underestimating the true prevalence in all animals surveyed. The high level of S. japonicum prevalence in goats indicates that they are likely important reservoirs in schistosomiasis transmission, necessitating their inclusion as targets of control, if the goal of elimination is to be achieved in China.

The current World Health Organization strategic plan targets the elimination of schistosomiasis as a public health problem by 2025 and accurate diagnostics will play a pivotal role in achieving this goal. DNA-based detection methods provide a viable alternative to some of the commonly used tests, notably microscopy and serology, for the diagnosis of schistosomiasis. The detection of parasite cell-free DNA in different clinical samples is a recent valuable advance, which provides significant benefits for accurate disease diagnosis. Here we validated a novel duplex droplet digital PCR assay for the diagnosis of Chinese (SjC) and Philippine (SjP) strains of Schistosoma japonicum infection in a mouse model. The assay proved applicable for both SjC and SjP infections and capable of detecting infection at a very early intra-mammalian stage in conveniently obtainable samples (urine and saliva) as well as in serum and feces. The target DNA copy numbers obtained in the assay showed a positive correlation with the infection burden assessed by direct traditional parasitology. The potential to detect parasite DNA in urine and saliva has important practical implications for large-scale epidemiological screening programmes in the future, particularly in terms of logistical convenience, and the assay has the potential to be a valuable additional tool for the diagnosis of schistosomiasis japonica.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a major antioxidant enzyme, which plays unique roles in the protection of cells against oxidative stress by catalysing reduction of lipid hydroperoxides. We isolated and characterized a full-length cDNA sequence encoding GPx gene from a blood fluke, Schistosoma japonicum (designated SjGPx), which contained an in-frame TGA codon for selenocysteine (Sec) and a concurrent Sec insertion sequence in its 3′-untranslated region. Protein encoded by SjGPx demonstrated a primary structure characteristic to the PHGPx family, including preservation of catalytic domains and absence of the subunit interaction domains. Semi-quantitative reverse transcription PCR and Western blotting showed that the SjGPx was mainly expressed in the female adults and eggs. RNA interference approach was employed to investigate the effects of knockdown of SjGPx. SjGPx expression level was significantly reduced on the 5th day post-RNAi. Significantly reduction in GPx enzyme activities, as well as obvious changes in morphology of intrauterine eggs followed the reduction in SjGPx transcript level. We observed a 63·04% reduction in GPx activity and the eggs severely deformed. Our results revealed that SjGPx protein might be involved in the provision of enzyme activity during egg production.

Schistosoma japonicum, a human blood fluke, causes a parasitic disease affecting millions of people in Asia. Thioredoxin–glutathione system of S. japonicum plays a critical role in maintaining the redox balance in parasite, which is a potential target for development of novel antischistosomal agents. Here we cloned the gene of S. japonicum thioredoxin (SjTrx), expressed and purified the recombinant SjTrx in Escherichia coli. Functional assay shows that SjTrx catalyses the dithiothreitol (DTT) reduction of insulin disulphide bonds. The coupling assay of SjTrx with its endogenous reductase, thioredoxin glutathione reductase from S. japonicum (SjTGR), supports its biological function to maintain the redox homeostasis in the cell. Furthermore, the crystal structure of SjTrx in the oxidized state was determined at 2·0 Å resolution, revealing a typical architecture of thioredoxin fold. The structural information of SjTrx provides us important clues for understanding the maintenance function of redox homeostasis in S. japonicum and pathogenesis of this chronic disease.

Schistosomiasis is one of the most prevalent, insidious and serious of the tropical parasitic diseases. Although the effective anthelmintic drug, praziquantel, is widely available and cheap, it does not protect against re-infection, drug-resistant schistosome may evolve and mass drug administration programmes based around praziquantel are probably unsustainable long term. Whereas protective anti-schistosome vaccines are not yet available, the zoonotic nature of Schistosoma japonicum provides a novel approach for developing a transmission-blocking veterinary vaccine in domestic animals, especially bovines, which are major reservoir hosts, being responsible for up to 90% of environmental egg contamination in China and the Philippines. However, a greater knowledge of schistosome immunology is required to understand the processes associated with anti-schistosome protective immunity and to reinforce the rationale for vaccine development against schistosomiasis japonica. Importantly as well, improved diagnostic tests, with high specificity and sensitivity, which are simple, rapid and able to diagnose light S. japonicum infections, are required to determine the extent of transmission interruption and the complete elimination of schistosomiasis following control efforts. This article discusses aspects of the host immune response in schistosomiasis, the current status of vaccine development against S. japonicum and reviews approaches for diagnosing and detecting schistosome infections in mammalian hosts.

The role that animals play in the transmission of Schistosoma japonicum to humans in the Philippines remains uncertain and prior studies have not included several species, adjustment for misclassification error and clustering, or used a cohort design. A cohort study of 2468 people providing stool samples at 12 months following praziquantel treatment in 50 villages of Western Samar, the Philippines, was conducted. Stool samples from dogs, cats, rats, and water buffaloes were collected at baseline (2003–2004) and follow-up (2005). Latent-class hierarchical Bayesian log-binomial models adjusting for misclassification errors in diagnostic tests were used. The village-level baseline and follow-up prevalences of cat, dog, and rat S. japonicum infection were associated with the 12-month cumulative incidence of human S. japonicum infection, with similar magnitude and precision of effect, but correlation between infection levels made it difficult to divide their respective effects. The cumulative incidence ratios associated with a 1% increase in the prevalence of infection in dogs at baseline and in rats at follow-up were 1·04 [95% Bayesian credible interval (BCI) 1·02–1·07] and 1·02 (95% BCI 1·01–1·04), respectively, when both species were entered in the model. Dogs appear to play a role in human schistosomiasis infection while rats could be used as schistosomiasis sentinels.

Serine protease inhibitors (serpin) play essential roles in many organisms. Mammalian serpins regulate the blood coagulation, fibrinolysis, inflammation and complement activation pathways. In parasitic helminths, serpins are less well characterized, but may also be involved in evasion of the host immune response. In this study, a Schistosoma japonicum serpin (SjB10), containing a 1212 bp open reading frame (ORF), was cloned, expressed and functionally characterized. Sequence analysis, comparative modelling and structural-based alignment revealed that SjB10 contains the essential structural motifs and consensus secondary structures of inhibitory serpins. Transcriptional profiling demonstrated that SjB10 is expressed in adult males, schistosomula and eggs but particularly in the cercariae, suggesting a possible role in cercarial penetration of mammalian host skin. Recombinant SjB10 (rSjB10) inhibited pancreatic elastase (PE) in a dose-dependent manner. rSjB10 was recognized strongly by experimentally infected rat sera indicating that native SjB10 is released into host tissue and induces an immune response. By immunochemistry, SjB10 localized in the S. japonicum adult foregut and extra-embryonic layer of the egg. This study provides a comprehensive demonstration of sequence and structural-based analysis of a functional S. japonicum serpin. Furthermore, our findings suggest that SjB10 may be associated with important functional roles in S. japonicum particularly in host-parasite interactions.

To detect the expression of pro-fibrotic molecules, such as heat shock protein 47 (Hsp47), transforming growth factor-beta 1 (TGF-β1) and connective tissue growth factor (CTGF) in liver specimens, and analyse their correlations with the progression of schistosomal hepatic fibrosis, liver biopsy was performed in 42 chronic schistosomiasis (CS) patients, 16 chronic hepatitis B (CHB) patients and five healthy individuals (HI). Immunohistochemistry (IHC) analyses displayed that the expression of Hsp47, TGF-β1 and CTGF was increased in CS and CHB patients compared with HI. Using real-time PCR, the mRNA levels of Hsp47, TGF-β1 and CTGF were higher in CS patients compared with HI. In CS patients, the mRNA levels of these genes were correlated with the stage of fibrosis, and TGF-β1 mRNA expression was associated with the grade of inflammation. Additional analyses indicated that the mRNA levels of Hsp47 and CTGF were highly correlated with liver stiffness value and spleen thickness diameter, both of which represented the severity of fibrosis. In conclusion, the three molecules are involved in the pathogenesis of hepatic fibrosis infected by Schistosoma japonicum. TGF-β1 participates not only in the inflammatory process, but also in the fibrotic process in which Hsp47 and CTGF probably play a key role.

As the currently only available molluscicide, niclosamide has been widely used for snail control for over 2 decades in China. There is therefore a concern about the emergence of niclosamide-resistant snail populations following repeated, extensive use of the chemical. The purpose of this study was to investigate the likelihood of niclosamide resistance in Oncomelania hupensis in China. Active adult O. hupensis snails derived from 20 counties of 10 schistosomiasis-endemic provinces of China, of 10 snails in each drug concentration, were immersed in solutions of 1, 0·5, 0·25, 0·125, 0·063, 0·032, 0·016 and 0·008 mg L−1 of a 50% wettable powder of niclosamide ethanolamine salt (WPN) for 24 and 48 h at 25 °C, and the median lethal concentration (LC50) was estimated. Then, the 24- and 48-h WPN LC50 values were compared with those determined in the same sampling sites in 2002. The results indicated that the 24- and 48-h WPN LC50 values for O. hupensis were not significantly different from those determined in 2002 (P = 0·202 and 0·796, respectively). It is concluded that the current sensitivity of O. hupensis to niclosamide has not changed after more than 2 decades of repeated, extensive application in the main endemic foci of China, and there is no evidence of resistance to niclosamide detected in O. hupensis.

Circulating microRNAs (miRNAs) have received considerable attention as a novel class of biomarkers for the diagnosis of cancer and as signalling molecules in mediating intercellular communication. Schistosomes, the causative agents of schistosomiasis, live in the blood vessels of a mammalian host in the adult stage. In the present study, we characterized schistosome-specific small RNA populations in the plasma of rabbits infected with Schistosoma japonicum (S. japonicum) using a deep sequencing method and then identified five schistosome-specific miRNAs, including four known miRNAs (Bantam, miR-3479, miR-10 and miR-3096), and one novel miRNA (miR-0001, miRBase ID: sja-miR-8185). Four of the five schistosome-specific miRNAs were also detected by real-time RT–PCR in the plasma of S. japonicum-infected mice. In addition, our study indicated that schistosome Argonaute 2/3 may be an excretory-secretory (ES) protein. In summary, our findings are expected to provide useful information for further development of novel biomarkers for the diagnosis of schistosomiasis and also for deeper understanding of the mechanism of host–parasite interaction.

Blockage of pathogen transmission through water decontamination is considered an important strategy for the prevention of schistosome infection. Many believe that this strategy is feasible, but it has yet to be achieved. Silver has a long history of use as a disinfectant. With the emergence of nanotechnology, silver can be shaped into nanoparticles which have been found to possess superb antimicrobial activities. In this light, we investigated the effects of silver nanoparticles (AgNPs) on Schistosoma japonicum cercariae. AgNPs rapidly induced cercarial tail-shedding, agitated behaviour and a decrease in cercarial secretion in a dose-dependent manner. Prolonged treatment was found to be cercariocidal, which nevertheless might be attributable to AgNP-induced cercarial tail loss rather than to toxicity. Higher concentrations of AgNPs (125 μg mL−1 and above) completely blocked cercarial infectivity. Despite decreased infectivity, cercariae exposed to lower concentrations of AgNPs for 30 min were still found capable of infecting hosts even without their tails, suggesting that tail loss does not necessarily signify a total loss of infective ability. We also found that silver ions (Ag+) were heavily involved in the observed cercarial responses of AgNPs. Our observations provide insight into the interactions between the larvae of helminth parasites and nanoparticles.

The aim of this study was to evaluate the therapeutic effects of osteopontin neutralization treatment on schistosome-induced liver injury in BALB/C mice. We randomly divided 100 BALB/C mice into groups A, B, C, D and group E. Mice in all groups except group A were abdominally infected with schistosomal cercariae to induce a schistosomal hepatopathological model. Mice in group C, D and group E were respectively administered with praziquantel, praziquantel plus colchicine and praziquantel plus neutralizing osteopontin antibody. We extracted mouse liver tissues at 3 and 9 weeks after the ‘stool-eggs-positive’ day, observed liver histopathological changes by haematoxylin-eosin and Masson trichrome staining and detected the expression of osteopontin, alpha-smooth muscle actin (α-SMA) and transforming growth factor-beta (TGF-β1) by immunohistochemistry, RT-PCR and Western blot. We found that praziquantel plus neutralizing osteopontin antibody treatment significantly decreased the granuloma dimension, the percentage of collagen and the expression of osteopontin, α-SMA and TGF-β1 compared to praziquantel plus colchicine treatment in both the acute and chronic stage of schistosomal liver damage (P<0·05). So we believe that the combined regimen of osteopontin immunoneutralization and anti-helminthic treatment can reduce the granulomatous response and liver fibrosis during the schistosomal hepatopathologic course.

A traditional assumption is that schistosome cercariae lose their tails at the onset of penetration. It has, however, recently been demonstrated that, for Schistosoma mansoni, cercarial tails were not invariably being shed as penetration took place and a high proportion of tails entered human skin under experimental conditions. This phenomenon was termed delayed tail loss (DTL). In this paper, we report that DTL also happens with S. japonicum cercariae during penetration of mouse skin. It occurred at all cercarial densities tested, from as few as 10 cercariae/2·25 cm2 of mouse skin up to 200 cercariae. Furthermore, it was demonstrated that there was a density-dependent increase in DTL as cercarial densities increased. No such density-dependent enhancement was shown for percentage attachment over the same cercarial density range.

Interleukin (IL)-13 and alternatively activated macrophages (AAMs) play an important role in liver granuloma and fibrosis of schistosomiasis. Paeoniflorin (PAE, C23H28O11) has been reported to have an anti-hepatic fibrosis effect in schistosomiasis; however, the mechanism has not been fully elucidated. In this study, we measured serum hyaluronic acid (HA) concentrations, liver granuloma diameter and volume density, fibrosis degree and expressions of IL-13, arginase-1 (ARG-1), nitric oxide synthase-2 (NOS-2), and phosphorylated signal transducer and activator of transcription 6 (p-STAT6) in mice liver of schistosomiasis. Then we detected expressions of specific biomarkers of AAMs and activity of Arg-1 in Kupffer cells (KCs) from infected and PAE-treated mice, or in KCs from uninfected mice, but exposed to rIL-13 in vitro. Finally, we observed expression of IL-13 signalling molecules in KCs and secretion of IL-13 from lymphocytes of infected and PAE-treated mice. Our results showed that during schistosomiasis, IL-13 expression and secretion increased with liver macrophages activated alternatively. PAE not only directly inhibited alternative activation of macrophages via reducing the phosphorylations of janus-activated kinase 2 (JAK2) and/or STAT6, leading to reduction of AAMs-related markers and Arg-1 activity, but also indirectly suppressed alternative activation of macrophages through decreasing secretion of IL-13. PAE might be a promising prophylactic agent for hepatic granuloma and fibrosis of schistosomiasis japonica.

In order to investigate the dynamics of Septin4 (Sept4) expression and its function in the formation of fibrotic livers in mice infected with Schistosoma japonicum, we constructed the mouse model of S. japonicum egg-induced liver fibrosis for 24 weeks. Immunohistochemical staining, qRT-PCR and Western blot were used to detect the expression of Sept4 and α-smooth muscle actin (α-SMA). We found Sept4 localized in the perisinusoidal space where hepatic stellate cells (HSCs) distribute in the periphery of circumoval granulomas and the portal venule. The expression of Sept4 and α-SMA had a similar significant tendency of an up-regulation to a peak at 12 weeks post-infection (p.i.) followed by a down-regulation. At 24 weeks p.i. both were at a low level. These results suggest that Sept4 and α-SMA may interact together in HSCs. Based on this evidence, we hypothesize that Sept4 seems to be involved in the formation of inflammatory granulomata and subsequent liver fibrosis by regulating HSCs activation.

Praziquantel is widely used for the treatment of human schistosomiasis. However, in recent years, there has been increasing concern about the resistance of Schistosoma species to praziquantel. The study described here was designed to evaluate the current susceptibility to praziquantel in S. japonicum in China. During the non-transmission period of schistosomiasis, a random sample of 4760 subjects from the main endemic foci of China were examined using parasitological stool examination. In total, 584 subjects were identified as being infected with S. japonicum, with a prevalence rate of 12 27%. Among them, 565 stool-egg-positive subjects were treated with praziquantel in a single oral dose of 40 mg/kg. Six weeks post-treatment, among the 505 villagers re-examined, 480 (95 05%) had no detectable S. japonicum eggs. Twenty-one subjects still excreting eggs after the first treatment were treated with praziquantel for the second time. All stool samples, including those from those participants with second treatment were re-examined 6 weeks after the second treatment, and no stool-egg-positives were found. The results indicate that the current efficacy of praziquantel against S. japonicum is still high and has not changed after more than 2 decades of repeated, expanded chemotherapy in the main endemic areas of China. It is suggested that no evidence of tolerance or resistance to praziquantel in S. japonicum was detected in China.

Schistosoma japonicum remains highly endemic in many counties in China and has recently re-emerged, to a large extent, in previously controlled areas. To test the hypothesis that small rodents and less agriculturally important domestic animals such as dogs and cats may play an important role in the transmission and potential re-emergence of this disease, an annual investigation of S. japonicum among humans, domestic animals and rodents, combined with detailed surveys of the snail intermediate host, was performed across 3 marshland villages and 3 hilly villages in Anhui province of China over 2 consecutive years. The highest infection prevalence and intensity observed across all mammals was in rodents in the hilly region; while in the marshland, bovines were suspected as the main reservoirs. However, relatively high infection prevalence levels were also found in dogs and cats in both regions. Such results may have implications for the current human- and bovine-oriented control policy for this medically and veterinarily important disease, particularly within the hilly regions of mainland China.