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Objective: To evaluate the application effect of Oncomelania hupensis snail crusher in batch detection., Methods: The O. hupensis snail crushers and triangular flasks for cercariae shedding were made. The uninfected snails were divided into 4 groups with the snail number of 50, 100, 200, 300, respectively, and then they were put in 40 triangular flasks, 10 flasks each group. Three flasks in each group were randomly selected, and 12 schistosome infected O. hupensis snails were put into the flasks (1 snail per flask). The 12 flasks with infected O. hupensis snails were put back to each group and were numbered uniformly. The O. hupensis snails in each group were crushed by the O. hupensis snail crushers, and the crushing rate of snails, and average crushing time and duration were recorded. When all the O. hupensis snails were crushed, water was added into the flasks, and water films from each flask were taken and observed under microscopes, the number of cercariae was recorded and the detection rates of each group were calculated. Meanwhile, the O. hupensis snails in the field were collected and detected by the crushing method and crushing and shedding method with O. hupensis snail crushers, and the coincidence rate of the two methods was calculated., Results: The crushing rate of snails in all the 4 groups were 100% , the average crushing times of groups with 50, 100, 200, 300 snails were 15.70, 23.20, 32.20, 39.20, respectively, and the average duration was 1.01, 1.70, 2.00, 3.00 min, respectively. Both the crushing time and duration were positively correlated with the number of snails detected (r = 0.68, 0.73, both P <0.01). The detection rates of cercariae in all the 4 groups were 100%. In the field application, 109 snails were detected by the crushing method, 620 ones were detected by the crushing and shedding method with O. hupensis snail crushers, and no infected snails were found, the coincidence rate of the two methods were 100%., Conclusion: The O. hupensis snail crusher with the advances of easy operation and labor-saving is valuable of field promotion.

Objective: To determine the viability of Schistosoma japonicum cercariae by staining., Methods: Schistosoma japonicum cercariae were stained by 0.4% trypan blue, 0.5% methylene blue-eosin-borax (M.E.B), 0.5% eosin, 0.5% methylene blue and 0.05% neutral red, respectively, for 5 min, then they were observed under a stereoscopic microscope., Results: The dead cercariae were stained in the trypan blue, M.E.B, eosin and neutral red, but unstained in the methylene blue. The vital cercariae were unstained in all the five kinds of dyes., Conclusion: The staining methods by using 0.4% trypan blue, 0.5%M.E.B, 0.5% eosin and 0.05% neutral red can be used to determine the viability of S. japonicum cercariae.

Objective: To study the morphological change of Schistosoma japonicum eggs processed by calcium cyanamide synthetic drug, so as to provide the basis for further study of the mechanism that calcium cyanamide synthetic drug to schistosome eggs., Methods: The calcium cyanamide synthetic drug was added to the cattle feces containing schistosome eggs and mixed up, and then the cattle feces was stacked as original shape on the marshland. Blank controls were set at the same time. The cattle feces samples were collected and.the schistosome eggs were observed under a microscope on the 1st, 2nd, 3rd, 7th day after the experiment., Results: By the effect of calcium cyanamide synthetic drug, the color of eggs was deepening gradually, the miracidia were atrophied, and the shells of eggs were thickened. The embryonic membrane of miracidia was no longer completed 3 days later, and the miracidia were deformed severely 7 days later. The atrophy of miracidia was not obvious in the blank controls., Conclusion: The schistosome miracidia and embryonic membrane can be damaged by the calcium cyanamide synthetic drug, and worse damaged with time extending.

Objective: To study the distribution characteristics of deposited eggs and pathological changes in the viscera of animal infected with Schistosoma japonicum at different time., Methods: New Zealand white rabbits were infected artificially with quantitative S. japonicum miracidia, then the distribution characteristics and the hatchability of schistosome eggs as well as the pathological changes of the corresponding viscera of the rabbits 42 and 60 d post-infection were observed and compared., Results: On the 42nd day post-infection, among all the viscera observed, the percentage of eggs deposited, the number of eggs per gram and the hatchability were the highest in the liver, while on the 60th day post-infection, the tissues and organs with the highest values of the above 3 indexes were the liver, rectum and upper section of the small intestine, respectively. From 42 day to 60 day post-infection, the liver of infected rabbits became swelling, hardening and lost elasticity, the color changed from black to dark grey, and egg nodules gradually appeared in the different sections of the small intestine, and also the mucosal hyperemia, edema and egg nodules were seen in the colon, cecum and rectum. The lesion levels tended to be correlated with the deposition of eggs., Conclusion: The amount and the density as well as the hatching rate of deposited eggs of S. japonicum in the viscera of infected rabbits at different time are different, and the lesion level in the host is correlated with the deposition of eggs.

Objective: To explore the enrichment technique of Schistosoma japonicum cercariae on the water surface, so as to establish a new method combined with the existing technology to detect the cercarial infested water body quickly and sensitively., Methods: Soybean oil, gasoline, kerosene and isophorone were screened as expanding agents. The cercariae were enriched by the thrust of the expanding agents when diffusing on the water surface, and PE adsorption film and C-6 film were applied to seize them so as to determine the infectivity of the water quickly. The relationship between the dose of expanding agents and diffusion radius were explored., Results: Gasoline, kerosene and isophorone were suitable expanding agents, and the diffusion effect of isophorone was the best. After the enrichment by the expanding agents, the detection rate of cercariae of the method seizing cercariae with the film significantly improved in the water., Conclusion: This new method could effectively improve the detection rate of the cercarial infested water and is suitable for the low-degree infested water.

A series of chiral praziquantel analogues were synthesized and evaluated against Schistosoma japonicum both in vitro and in vivo. All compounds exhibited low to considerable good activity in vivo. Remarkably, worm reduction rate of R-3 was 60.0% at a single oral dose of 200mg/kg against juvenile stage of Schistosoma japonicum. The target compounds displayed in vivo antischistosomal activity against both Schistosoma japonicum and Schistosoma mansoni. Furthermore, all R-isomers displayed stronger antischistosomal activity than S-isomers in vivo, indicating R-isomers were the active enantiomers, while S-isomers were less active ones. This structure activity relationship (SAR) could have important implications in further drug development for schistosomiasis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Allograft inflammatory factor-1 (AIF-1) plays an important role in various inflammatory conditions. Our previous study demonstrated that AIF-1 was over-expressed in the liver of BALB/c mice infected with Schistosoma japonicum and played significant role in the pathogenesis of schistosomiasis. The aim of this study was to focus on the effect of AIF-1 treatment on liver fibrosis and necrosis of BALB/c mice infected with S. japonicum. Seventy-two BALB/c mice were infected with cercariae of S. japonicum and then divided into three groups: AIF-1-treated group, saline-treated group, and control group. The vital signs, liver function, egg load, and hepatic pathological changes of the mice were assessed, and the levels of AIF-1 and TNF-α in the liver and spleen were measured at 5, 8, and 14 weeks postinfection. The treatment of AIF-1 on the mice infected with S. japonicum suppressed the expression of TNF-α and increased the effectiveness of AIF-1 in the liver and spleen at 14 weeks postinfection. Histopathological analysis and Masson trichrome staining for the liver tissues showed that the liver fibrosis and necrosis were alleviated previously compared with other infected mice at 14 weeks postinfection. The treatment of AIF-1 on the mice infected with S. japonicum can alleviate hepatic fibrosis and necrosis which indicate that AIF-1 use may prevent and cure the liver fibrosis.

Microtus fortis is a non-permissive host for Schistosoma japonicum. While M. fortis lymphocytes are known to provide natural resistance against S. japonicum, the specific mechanism remains unclear. A bone marrow transplantation (BMT) model was established using immunodeficient mice, either nude (experiment 1) or V(D)J recombination activation gene deficient mice (RAG-1(-/-)) (experiment 2) as recipients and M. fortis or C57BL/6 mice as donors. The growth and development of S. japonicum were evaluated in each group to assess the role of M. fortis lymphocytes in the response to infection. Lymphocyte ratios and S. japonicum-specific antibody production in transplanted groups increased significantly compared to those in non-transplanted group. Spleen indices and density of splenic lymphocytes in transplanted RAG-1(-/-) mice were higher than those in non-transplanted RAG-1(-/-) mice. No difference in the worm burden was observed among group A (transplants derived from M. fortis), B (transplants derived from C57BL/6 mouse) and C (non-transplanted mice), although worms in group A were shorter than those in other groups, except non-transplanted RAG-1(-/-) mice. Reproductive systems of worms in mice (nude or RAG-1(-/-)) transplanted from M. fortis were not as mature as those in mice (nude or RAG-1(-/-)) transplanted from C57BL/6 mouse and non-transplanted nude mice, but they were more mature than worms in non-transplanted RAG-1(-/-) mice. Therefore, the transplantation model using nude and RAG-1(-/-) mice was successfully established. The M. fortis lymphocytes did not appear to affect the S. japonicum worm burden, but they led to schistosome shortening and a significant reduction in parasite spawning. Thus, M. fortis cellular and humoral immunity provides a defense against schistosomes by negatively impacting the parasite growth and reproductive development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To clone and express a high mobility group box 1(HMGB1) protein of Schistosomajaponicum (Mainland strain) and analyze its function., Methods: The DNA fragment of open reading frame encoding Sj HMGB 1 protein was amplified by RT-PCR from the mRNA of S. japonicum worms, then it was subcloned into the expression vector pET28a(+) to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3), and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombinant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding capacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 protein and infected with 45 +/- 2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection, the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue, respectively. The worm and egg reduction rates were calculated respectively., Results: A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR, which was the open reading frame (ORF) encoding SjHMGBlprotein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA, and the recombinant protein immunized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abundantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effective immune protection against S. japonicum., Conclusion: The gene encoding HMGB1 from S. japonicum and the soluble recombinant SjHMGB1 protein with natural functional activity are obtained, and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

Objective: To prokaryotically express the valosin-containing protein (VCP) of Schistosoma japonicum, and analyze its VCP mRNA expressions in the cercaria, schistosomulum, adult worm (female and male worms) and egg., Methods: RNA of S. japonicum eggs were extracted, and reversely transcribed into cDNA. The VCP gene of S. japonicum was amplified by using polymerase chain reaction (PCR), and subcloned into the prokaryotically expressed vector pET15b. The recombined plasmid was transformed into BL21 cells, and the expression of the target gene was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). The recombinant protein was yielded through the purification of inclusion body, and identified by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The RNA (s) of cercaria, schistosomulum, female adult worm, male adult worm, and egg of S. japonicum were extracted, digested with DNase, purified, and reversely transcribed into cDNA. The mRNA expressions of the VCP gene in various developmental stages of S. japonicum were determined by using fluorescence-based quantitative real-time PCR., Results: The VCP gene of S. japonicum was yielded by PCR amplification, and the recombinant protein was obtained through recombinant plasmid expression and purification of inclusion body. The highest VCP mRNA expression in S. japonicum cercaria was detected by the fluorescence-based quantitative real-time PCR, while low expressions were found in the schistosomulum, egg, female and male adult worms., Conclusion: The recombinant protein encoded by the VCP gene of S. japonicum is successfully obtained, and the VCP mRNA expression is determined in various developmental stages of S. japonicum.

In order to explore the interaction of mefloquine with hemin against adult Schistosoma japonicum in vitro, the 50% and 95% lethal concentration (LC50 and LC95) of mefloquine and hemin against schistosomes, some factors, such as other iron providing agents, iron chelaters, zinc protoporphyrin-IX, and biological relevant reductants, that might impact on antischistosomal activity induced by interaction of mefloquine with hemin, and preliminary analysis of chemical interaction of both compounds were undertaken. The LC50 and LC95 of mefloquine and hemin alone against schistosomes were determined to be 6.5μg/ml and 7.8μg/ml as well as 232μg/ml and 355μg/ml, respectively. The LC50 and LC95 of mefloquine in the presence of hemin 100μg/ml was 0.24μg/ml and 0.59μg/ml, respectively. On the other hand the LC50 and LC95 of hemin in the presence of mefloquine 1μg/ml was 23.2μg/ml and 77.2μg/ml, respectively. Meanwhile, mefloquine/hemin combinations showed potential synergistic effects against adult S. japonicum, with combination index (CI) values <1. Apart from hemin, zinc protoporphyrin-IX, and other iron providing agents such as ferrous sulfate and ferriammonium sulfate combined with mefloquine exhibited no toxic effect against schistosomes. On the other hand, addition of iron chelators (deferiprone, desferrioxamine mesylate, or 2,2'-bipyridine) to the medium containing mefloquine-hemin resulted in no protective effect on the worms. Furthermore, biological reductants like glutathione, vitamine C or cysteine showed no apparent worm protection effect from toxic mefloquine-hemin even at higher concentrations (242.3-614.6μg/ml, i.e., 6.4-17.8-fold higher than the concentration of hemin). Chemical interaction of mefloquine with hemin was studied in 40% DMSO-Tris buffer solution. Both UV-Vis spectrum and mass spectrum demonstrated the strong interaction of mefloquine with hemin, which resulted in a reduction of hemin color and emergence of an adduct formed by mefloquine and hemin. The results confirm that mefloquine combined with hemin exhibits potential synergistic effect against adult S. japonicum in vitro., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Pairing of Schistosoma japonicum initiates female development, leads to female sexual maturation, and maintains this mature state. To understand the mechanism involved in these processes, we studied parasites isolated from single- and double-sex cercariae-infected mice using deep-sequencing analysis, Solexa, to uncover pair-regulated transcriptional profiles. In this study, we report the results of high-throughput tag-sequencing (Tag-seq) analysis of the transcriptome of female worms 18 and 23 days postsingle- and double-sex infections. We sequenced over 3 million tags, obtained a total of 14,034, 27,251, 22,755, and 22,555 distinct tags corresponding to 5,773, 9,794, 8,885, and 8,870 tag-mapped genes for 23-day-old female schistosomula from double-sex infections (23DSI), 23-day-old female schistosomula from single-sex infections (23SSI), 18-day-old female schistosomula from double-sex infections (18DSI), and 18-day-old female schistosomula from single-sex infections (18SSI), respectively. Analyses of differentially expressed genes revealed similarities in the gene expression profiles between 18SSI and 18DSI as well as rational differential gene expression between 18SSI and 23SSI. However, fewer upregulated genes were found in 23DSI compared with 18DSI. Of the 3,446 differentially expressed genes between 23DSI and 23SSI, 2,913 genes were upregulated in 23SSI, whereas only 533 genes were upregulated in 23DSI. In these upregulated genes in 23DSI, phosphoglycerate mutase, superoxide dismutase, egg antigen, ribosomal proteins, ferritin-1 heavy chain, and eukaryotic translation initiation factor 2 were detected. Detection of these genes suggests that gene expression in 23DSI is specialized for functions such as promotion and maintenance of female sexual maturation and egg production. Quantitative real-time (RT)-PCR analysis confirmed the Solexa results, thereby supporting the reliability of the system. Our results offer new insights into the biological significance of pairing, which directs the expression of genes specific for sexual maturation and egg production.

Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have reemerged, and to characterize infrapopulations in individual hosts. The life stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4 years), considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with the discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.

Objective: To explore the optimizational time of artificial population schistosome infected Oncomelania hupensis snails., Methods: Under laboratory conditions, the snails were infected with the miracidia of Schistosoma japonicum for 2 h, 3 h and 4 h respectively, and the death rates and the infection rates of the snails, and the quantities of cercariae of each group were observed 60-120 d after the infection, and all the data observed were analyzed to get the optimizational time of artificial population schistosome infected snails., Results: Of the 3 h group, the snail infection rate was the highest and the mortality was the lowest among the 3 groups (P<0.05). The average number of cercariae of the 3 h group was higher than that of the 2 h group (P<0.05), while there was no statistical difference between the 3 h group and the 4h group (P>0.05)., Conclusion: Under laboratory conditions, the optimizational time is 3 h in artificial population schistosome infected O. hupensis snails.

The interplay between sexes is a prerequisite for female growth, reproductive maturation, and egg production, and the basis of schistosome pathopoiesis and propagation. The tegument is in direct contact with the host environment and its surface membranes are particularly crucial for schistosome survival in the definitive host. In this study, a streptavidin-biotin affinity purification technique combined with LC-MS/MS was used to analyze putative tegument-exposed proteins in female and male adult Schistosoma japonicum worms. In total, 179 proteins were identified in females and 300 in males, including 119 proteins common to both sexes, and 60 female biased and 181 male biased proteins. Some (e.g., serpin and CD36-like class B scavenger receptor) were involved in host-schistosome interactions, while some (e.g., gynecophoral canal protein) were important in the interplay between sexes. Gene Ontology analysis revealed that proteins involved in protein glycosylation and lysosome were highly expressed in females, while proteins involved in intracellular signal transduction, regulation of actin filament polymerization, and proteasome core complex were highly expressed in males. These results might elucidate physiological differences between the sexes. Our study provides new insights into schistosome growth and sexual maturity in the final host and permits the screening of vaccine candidates or drug targets for schistosomiasis.

About 46 mammal species have been suspected as reservoir hosts for Schistosoma japonicum and therefore the track of the target parasites, in relation to definitive host species, may be of great importance in terms of theoretical and practical implications. The circadian rhythm of cercariae emergence, a genetically controlled behavior for parasites to adapt to their definitive hosts, may seem to be a perfect biological marker for S. japonicum. In this study, a late (or nocturnal) cercarial emergence pattern was observed on the parasites from one hilly region in Anhui of China, where rodents serve as reservoirs, and on the first generation of the parasites. Moreover, by using the circular statistics, the homogeneity of parasites in such trait was also demonstrated. All these provide evidence for the genetically controlled biological trait, which seems essential in the investigation of macro- or micro-dynamics of parasite transmission of interest. This is particularly true in the case of S. japonicum when multiple parasite isolates or strains are more likely to exist., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tsunagi/Y14 is an evolutionarily conserved RNA-binding protein that is required for the maintenance of oogenesis and the masculinization of the germ-line in many animal models. We speculated that Tsunagi/Y14 might also regulate reproductive organ development in Schistosoma japonicum (S. japonicum, Sj). Sj Tsunagi/Y14 and control double-stranded RNAs were introduced into schistosomula by electroporation respectively. These transfected schistosomula were cultured in vitro for 1, 3 or 5 days. The mRNA and protein levels of the target gene in the cultured schistosomula were significantly suppressed compared with those of the control group. Furthermore, BALB/c mice were infected with the transfected schistosomula for 6 weeks and were sacrificed to harvest the adult worms. We found that the silencing of Sj Tsunagi/Y14 led to defects in reproductive organs development in both male and female worms. Moreover, it also affected the size, quantity and activity of the eggs in the mice liver. Our findings indicated that Tsunagi/Y14 plays a critical role in the development of reproductive organs and eggs in S. japonicum., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To understand the pathological changes of tissues and organs in Microtus fortis infected with Schistosoma japonicum for further insight into the biological dynamic variation and explore immune mechanisms of M. fortis against S. japonicum infection., Methods: The healthy M. fortis and mouse were infected with cercariae of S. japonicum through abdominal skin. On the day 12, 20 and 40, the M. fortis and mouse were sacrificed by cervical dislocation and dissected. The liver and kidney slices of M. fortis and mouse of the different groups were prepared and stained with hematoxylin eosin stain (HE) by conventional techniques, respectively., Results: We did not find obvious lesions on the internal organs of mouse 12 d and 20 d after the infection and the lesions of M. fortis 40 d after the infection. However, white nodules appeared on the liver, kidney and spleen of the M. fortis 12 d and 20 d after the infection, and there were a lot of S. japonicum worms on pathological sections of inflamed liver and kidney tissues, and the boundary between worms and normal organization was clear. There were no parasites on pathological section of liver and kidney of mouse 12 d and 20 d after the infection., Conclusion: Among M. fortis, there are severe immune responses and the immune responses have an individual difference 12 days after S. japonicum infection.

Identification of high-risk regions of schistosomiasis is important for rational resource allocation and effective control strategies. We conducted the first study to apply the newly developed method of adaptive kernel density estimation (KDE)-based spatial relative risk function (sRRF) to detect the high-risk regions of schistosomiasis in the Guichi region of China and compared it with the fixed KDE-based sRRF. We found that the adaptive KDE-based sRRF had a better ability to depict the heterogeneity of risk regions, but was more sensitive to altering the user-defined smoothing parameters. Specifically, the impact of bandwidths on the estimated risk value and risk significance (P value) was higher for the adaptive KDE-based sRRF, but lower on the estimated risk variation standard error (s.e.) compared with the fixed KDE-based sRRF. Based on this application the adaptive and fixed KDE-based sRRF have their respective advantages and disadvantages and the joint application of the two approaches can warrant the best possible identification of high-risk subregions of diseases.

Schistosomiasis is a tropical disease, which could do serious damage to the people's health, and it hinders the development of the social economy but may be neglected. After a positive control, some countries and regions have blocked the spread of schistosomiasis. However, in the past few years, with the development of social economy, due to the global movement of people, schistosomiasis not only poses a threat to control areas, but also may cause new endemic areas. This article reviews the parasitological characteristics, clinical manifestations, epidemiological situation, and control approaches of three major kinds of human schistosomiasis, schistosomiasis japonica, schistosomiasis haematobia, and schistosomiasis mansoni.

Background: When compared to the murine permissive host of Schistosoma japonicum, Wistar rats are less susceptible to Schistosoma japonicum infection, and are considered to provide a less suitable microenvironment for parasite growth and development. MicroRNAs (miRNAs), are a class of endogenous, non-coding small RNAs, that impose an additional, highly significant, level of gene regulation within eukaryotes., Methods: To investigate the regulatory mechanisms provided by miRNA in the schistosome-infected rat model, we utilized a miRNA microarray to compare the progression of miRNA expression within different host tissues both before and 10 days after cercarial infection, in order to identify potential miRNAs with roles in responding to a schistosome infection., Results: Among the analysed miRNAs, 16 within the liver, 61 within the spleen and 10 within the lung, were differentially expressed in infected Wistar rats. Further analysis of the differentially expressed miRNAs revealed that many important signal pathways are triggered after infection with S. japonicum in Wistar rats. These include the signal transduction mechanisms associated with the Wnt and MAPK signaling pathways, cellular differentiation, with a particular emphasis on adipocyte and erythroid differentiation., Conclusions: The results presented here include the identification of specific differentially expressed miRNAs within the liver, lungs and spleen of Wistar rats. These results highlighted the function of host miRNA regulation during an active schistosome infection. Our study provides a better understanding of the regulatory role of miRNA in schistosome infection, and host-parasite interactions in a non-permissive host environment.

Schistosoma japonicum has a complex lifecycle and exhibits dramatic changes in its biology and morphology at different developmental stages. The schistosomulum and adult worm are two stages of this complex lifecycle and differentially expressed proteins in these two stages should be important for survival, development, and reproduction of the parasites. In this study, soluble and hydrophobic proteins were extracted from eggs, cercariae, schistosomula (8d and 19d), and male and female adult worms (42d) of Schistosoma japonicum, and separated by two-dimensional (2D) gel electrophoresis. A total of 1376±52, 928±61, 1465±41, 1230±30, 904±34, and 1080±26 soluble proteins and 1437±44, 845±53, 986±22, 1145±35, 1066±39, and 1123±45 hydrophobic proteins were separated from eggs, cercariae, schistosomula (8d and 19d), and male and female adult worms (42d), respectively. There were 65±14, 27±7, 37±17 and 48±9 soluble protein spots only present in schistosomula (8d and/or 19d) and adult schistosomes (male and/or female). We successfully identified 22 spots from schistosomula and 11 spots from adult schistosomes by mass spectrometry. Quantitative real-time RT-PCR was used to examine six differentially expressed proteins at the transcription level. These proteins only found in schistosomula or adults stage by the proteomics analysis were highly expressed in the corresponding stage at mRNA level. Bioinformatics analysis showed that the differentially expressed proteins from schistosomula were mainly involved in cellular metabolic processes, stress response and developmental process. Differentially expressed proteins from adult schistosomes were involved with gene expression and protein metabolism processes. The results of this study might provide new insights to stimulate further exploration of the mechanism of growth and development in schistosomes and help identify candidate molecules for developing new vaccines or drugs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Apoptosis is an important aspect of a number of biological processes, from embryogenesis to the stress-injury response. It plays a central role in balancing cell proliferation and tissue remodeling activity in many organisms. In the present study, apoptosis in 14 days post infection schistosomula was evaluated using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays and DAPI staining. Additionally, flow cytometry using the Annexin V-FITC/propidium iodide (PI) (Annexin V/PI) assay confirmed the percentage of early apoptotic, late apoptotic, and necrotic cells in 14 and 23 days post infection worms. Conserved Domain Database (CDD) BLAST analysis and alignment analysis of known schistosome proteins demonstrated the feasibility of detecting the activity of caspase-3 and -7 using the caspase-3/7 Glo analysis assay. Analysis of caspase-3 and -7 activities in schistosome demonstrated that both caspases were active in each developmental stage of Schistosoma japonicum, but was highest in the 14 days post infection schistosomula. Additionally, the caspase peptide inhibitor (Z-VAD-FMK) inhibited the caspase-3/7 activity at all developmental stages examined. Therefore, we hypothesized that two main signaling pathways are involved in apoptosis in S. japonicum, the caspase cascade and the mitochondrial-initiated pathway. We have constructed a model of these two pathways, including how they may interact and their biological outcomes. qRT-PCR analyses of the gene expression profiles of apoptosis-related genes supported our hypothesis of the relationship between the apoptotic pathway and parasite development. The data presented here demonstrates that apoptosis is an important biological process for the survival and development of the schistosome, and identifies potential novel therapeutic targets., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Aim: To investigate the effects of different concentrations of Schistosoma japonicum (S. japonicum) egg antigen on fibrogenesis and apoptosis in primary hepatic stellate cells (HSCs)., Methods: A mouse model of schistosomiasis-associated liver fibrosis (SSLF) was established by infecting mice with schistosomal cercaria via the abdomen. HSCs were isolated from SSLF mice by discontinuous density gradient centrifugation, and their identity was confirmed by immunofluorescence double staining of α-smooth muscle actin (α-SMA) and desmin. The growth inhibitory effect and 50% inhibitory concentration (IC50) of S. japonicum egg antigen for primary HSCs (24 h) were determined using a cell counting kit-8 (CCK-8) assay. The expression levels of α-SMA, matrix metalloproteinase-9 (MMOL/LP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in HSCs in response to different concentrations of S. japonicum egg antigen were detected by Western blotting and real-time reverse transcription-polymerase chain reaction. The levels of phospho-P38 (P-P38), phospho-Jun N-terminal kinase (P-JNK) and phospho-Akt (P-AKT) in HSCs were detected by Western blotting., Results: An SSLF mouse model was established, and primary HSCs were successfully isolated and cultured. S. japonicum egg antigen inhibited HSC proliferation in a concentration-dependent manner. The IC50 of the S. japonicum egg antigen was 244.53 ± 35.26 μg/mL. S. japonicum egg antigen enhanced α-SMA expression at both the mRNA and protein levels and enhanced TIMP-1 expression at the mRNA level in HSCs (P < 0.05), whereas the expression of MMOL/LP-9 was attenuated at both the mRNA and protein levels in a concentration-dependent manner (P < 0.05). A high concentration of S. japonicum egg antigen enhanced P-P38, P-JNK and P-AKT activation (P < 0.05). The changes in α-SMA and MMOL/LP-9 expression induced by S. japonicum egg antigen were closely correlated with P-P38 and P-JNK activation (P < 0.05). The attenuation of MMOL/LP-9 was also correlated with P-AKT activation (P < 0.05), but the increase in α-SMA expression was not. TIMP-1 expression was not correlated with P-P38, P-JNK or P-AKT activation., Conclusion: S. japonicum egg antigen promotes fibrogenesis, activates the P38/JNK mitogen-activated protein kinase and AKT/PI3K signaling pathways and inhibits proliferation in primary HSCs isolated from SSLF mice in a concentration-dependent manner.

Objective: To investigate the effect of Pulsatilla chinensis (Bunge) Regel saponins (PRS) against the eggs, miracidia, cercariae of Schistosoma japonicum in vitro and compare its efficacy with praziquantel., Methods: ICR mice were infected with the cercariae of S. japonicum by the patching abdominal method. The livers of the mice were grinded, screened, and then the eggs of S. japonicum were obtained 42 days post-infection. The miracidia were hatched by using the eggs, and the cercariae were obtained by using the infected Oncomelania snails on the light. The eggs, miracidia and cercariae of S. japonicum were incubated with 0, 1, 2, 3, 4, 5, 6, 7, 8 microg/ml PRS for different time, and praziquantel (PZQ) was used as the control., Results: PRS suppressed the hatching rates of eggs for 24 h slightly superior to that of the control drug PZQ at different concentrations, especially in the 4 microg/ml concentration. After the miracidia were incubated with 1, 2, 3, 4, 5, 6, 7, 8 microg/ml PRS for 30 min, the dead rates of miracidia were 13.47, 26.05, 60.99, 90.84, 100, 100, 100, 100%, respectively. After the cercariae were incubated with 1, 2, 3, 4, 5, 6, 7, 8 microg/ml PRS for 30 min, the dead rates of cercariae were 5.32, 18.81, 44.7, 76.87, 98.28, 100, 100, 100%, respectively. PRS showed time- and dose-dependent mortality effects on the miracidia and cercariae of S. japonicum., Conclusion: PRS has the effects against eggs, miracidia, cercariae of S. japonicum in vitro, and it may become a new anti-schistosome agent.

Schistosoma blood flukes, which cause schistosomiasis affecting 200 million people in the world, are dependent on signals from host CD4(+) T cells to facilitate parasite growth and development in the mammalian host and to induce Th2-biased inflammatory granulomas. B cells, however, are reported to down-regulate granulomatous pathology in schistosomiasis, but not to affect the development of blood flukes together with CD4(+) T lymphocytes. Thus it is not clear whether B cells mediate parasite development, reproduction and egg granuloma formation of schistosomes without the help of CD4(+) T lymphocytes. Using mice that have severe combined immunodeficiency (scid) and mice lacking T cells (nude), we found that the absence of B cells can more seriously hamper the development and paring of adult worms, but granuloma formation of Schistosoma japonicum in scid mice was not down-regulated comparing with that in nude mice. The level of IL-10 in the sera of nude mice was significantly higher than of scid mice at 43 days post infection (p.i.). Thus multiple mechanisms of immune modulation seem to be involved in parasite development and reproduction by helminth-induced regulatory B cells. Our findings have significance for understanding the molecular connections between schistosomes and T- and B-cells, indicating that more research is needed to develop efficient vaccine-based therapies for schistosomiasis.

Objective: To explore the infectivity difference between the mice challenged by laboratory-cultivated and field collected Schistosomajaponicum-infected Oncomelania hupensis snails., Methods: A total of 120 Kunming mice were randomly divided into two groups. S. japonicum-infected O. hupensis releasing cercariae was according to routine cercaria shedding method. Each mouse was challenged by 40 cercariae. The worm-load, the development rate, EPG of liver and EPG of feces in the mice were calculated., Results: The mean worm-load, adult worm development rate, EPG of liver and EPG of feces in the group of mice infected by field collected S. japonicum-infected snails were 27.43 +/- 3.78, 68.53 +/- 9.44, 19 800.97 +/- 6 752.59 and 196.37 +/- 11.56, respectively, which were significantly higher than those in the group challenged by cercariae from laboratory-cultivated S. japonicum-infected snails (23.93 +/- 4.93, 59.83 +/- 12.32, 5 803.69 +/-1 560.49 and 107.73 +/- 10.32) (P < 0.05). The mean worm-load, adult worm development rate and EPG of liver in the male mouse group were higher than those in the female mouse group (P < 0.05)., Conclusion: The cercariae released from field collected S. japonicum-infected snails have more aggressive infection ability, compared with the cercariae released from the laboratory-cultivated S. japonicum-infected snails and the results also show male mice are more susceptibility to the schistosome infection than female mice.

Objective: To evaluate the effect of agroforestry models on the inhibition of Oncomelania snails in the plateau mountainous area of Yunnan Province., Methods: The experimental field was established at Sanying Village of Eryuan County, Yunnan Province, where the "Flourishing Forest and Controlling Snails Project" was implemented. Different drought crops (alfalfa, vegetables, broad bean, garlic, lettuce, celery, green onions, and wheat) were intercropped under walnut forest in experimental groups, and the crops were not intercropped under walnut forest in a control group. The growth of forest, the change of snails and short-term income of residents were investigated., Results: Agroforestry models promoted the forestry growth and effectively inhibited the growth of snails. There was a little snail in one of the experimental group that forest was intercropped with alfalfa (the occurrence rate of frames with living snails was 3.33%, the average density of living snails was 0.004/0.1 m2, and the declining rates were both 50.00%). The snails were not found in other intercropped models. The income of residents in the experimental groups increased (900-6 800 Yuan per year) compared with that in the control group., Conclusion: The model of walnut forest intercropped with crops not only has the obvious effect on inhibition of snails, but also has good economic and ecological benefits in the plateau mountainous area of Yunnan Province.

Objective: To evaluate the killing effect of BTW5 on both juvenile and adult worms of Schistosoma japonicum in vitro., Methods: The mice were infected with cercariae of Schistosoma japonicum obtained from infected Oncomelania hupensis. The juvenile worms were obtained from the liver and mesenteric veins of the mice by perfusion 18 d after the infection, and the adult worms were obtained from the liver and mesenteric veins of the mice by perfusion 5 weeks after the infection. The adult and juvenile worms were cultivated in DMEM medium containing different concentrations of BTW5 and were observed for 3 d. After the observation, the worms were stained by hydrochloric acid carmine to observe the injuries., Results: The death rates and motility reducing rates of adult and juvenile worms in DMEM medium containing different concentrations of BTW5 were significantly higher than those in the controls. The staining suggested that BTW5 had a damage effect on the tegument and the intestinal canal of the male and female worms, in addition to the ovaries of the female worms., Conclusion: BTW5 has an antischistosomal effect on adult and juvenile worms of Oncomelania hupensis.

Rodent models for Schistosoma japonicum infections have demonstrated that these animals possess a degree of resistance to schistosome infections that may be both T and B lymphocyte-mediated. However, their exact role is not well-defined and other immune mechanisms are likely to also play a role in protecting against infection. Immunosuppressed and immunocompetent reed voles (Microtus fortis, Mf), rats and mice (n=24/group) were infected with S. japonicum, and animals were sacrificed 42 days later under anesthesia. Neither worms nor eggs were observed in infected immunosuppressed Mf or rats, with the exception of one rat that presented with few eggs. In immunosuppressed mice, changes in the number and size of the worms were not significantly different compared to immunocompetent mice, but worm fecundity was affected. The size and number of granulomas in immunosuppressed animals was also reduced. Analysis of serum antibodies specific to schistosome adult worm antigen at 3 weeks post-infection demonstrated that the levels of antibodies in the sera of rats were significantly higher than in Mf and mice. In addition, Mf serum levels of IL-4 and IL-12 were significantly higher than levels observed in rats and mice. Antibodies and cytokines in the sera of Mf peaked 3 weeks post-infection and then began to decrease, while antibody responses in rats and mice increased gradually between weeks 3-7 post-infection. It is possible that T and B cells have a dual role in both mediating protection and exacerbating disease outcomes., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Artesuante (AS) is a good chemoprophylactic drug for preventing against schistosome infection, but Hua et al. recently reported that the sensitivity of AS against Schistosoma japonicum decreased after 10 years of use in China. There are at least three problems, to our knowledge, making the finding suspicious or inconclusive in that report. In consideration of it as the first report about the emergence of potential artemisinin derivative-resistant S. japonicum to date and the possible severe influences of the emergence of AS-resistant S. japonicum on the future choice of chemoprophylactic drugs for preventing against S. japonicum infections in China, we write this comment and call for some more rigorous and convincing trials to confirm this finding further.

The aim of the present study is to further understand and analyze the interaction of mefloquine with praziquantel against adult Schistosoma japonicum in vitro. Mice infected with S. japonicum cercariae for 35-37 days were sacrificed, and adult schistosomes were collected by perfusion. Schistosomes were placed to each of 12 wells of a Falcon plate and maintained in RPMI 1640 supplemented by 10% calf serum. For determination of 50% and 95% lethal concentration (LC50 and LC95) of the two drugs in vitro, schistosomes were exposed to mefloquine at concentrations of 1, 2, 3, 4, 5, 6, 7, and 10 μg/mL or praziquantel at concentrations of 0.001, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 10, and 30 μg/mL. The plate was incubated at 37°C in 95% air + 5% CO₂ for 72 h. According to the half-life of oral mefloquine and praziquantel in mice, mefloquine combined with praziquantel simultaneously, mefloquine administered within 1 h after praziquantel and praziquantel administered within 17 h after mefloquine were used to evaluate the effect of mefloquine in combination with praziquantel against S. japonicum in vitro. The results showed that the LC50 and LC95 of mefloquine calculated by the Bliss method were 6.17 μg/mL (95% confidence limits, 5.84-6.517 μg/mL) and 8.703 μg/mL (95% confidence limits, 7.632-9.797 μg/mL), respectively. As to praziquantel, no worm death was seen when schistosomes were exposed to praziquantel at concentrations of 0.005-0.2 μg/mL for 72 h. While in the worms exposed to praziquantel 1, 10, and 30 μg/mL, strong spasmodic contractions of the worm body and vesiculation along the worm surface were observed, but 48-75% of the schistosomes survived the exposure in 72-h incubation. Meanwhile, the number of dead worms that emerged in each group was not proportion to the increasing concentrations. Therefore, it is not appropriate to calculate the LC50 and LC95 of praziquantel. For evaluation of the interaction with the two drugs, praziquantel 0.1 or 0.2 μg/mL, which may induce moderate or strong spasmodic contractions of the worm body and vesiculation along the worm surface, was combined with mefloquine 5, 6, or 7 μg/mL. It was found that when mefloquine combined with praziquantel simultaneously or administered 1 h after addition of praziquantel, the spasmodic contraction of the male worm body was antagonized by mefloquine in various degrees according to the concentrations of mefloquine used. Meanwhile, praziquantel-induced weakened motor activity could be reversed by mefloquine. In female worms, morphological alterations and stimulated motor activity induced by mefloquine still developed. Interestingly, using these two regimens to combine mefloquine with praziquantel resulted in no impact or a decrease in worm mortality. On the other hand, praziquantel 0.2 μg/mL administered within 17 h after mefloquine 5 or 6 μg/mL promoted the damage to the tegument of the worms, which led to enhance the worm mortality compared with that of worms exposed to mefloquine alone. The results indicate that in vitro higher concentrations of praziquantel administered within 17 h after mefloquine may increase the effect against adult schistosomes, while praziquantel combined with mefloquine simultaneously or administered 1 h before addition of mefloquine exhibits no impact or decrease in the effect against schistosomes.

Oncomelania hupensis is the intermediate host of Schistosoma japonicum. In the present study, we investigated the effects of protein extracts from head-foot or gland tissue of O. hupensis on mother sporocysts of S. japonicum cultured in vitro. In the presence of head-foot protein extract of snails from the native province Hunan, in-vitro-transformed mother sporocysts presented not only a longer survival time and stronger motility, but also a bigger size than parasites cultured with protein extracts of glands of the same snail or head-foot tissue of a non-native snail from the Hubei province. Using suppression subtractive hybridization, two subtractive libraries were constructed on the basis of RNA of sporocysts cultured with or without native snail head-foot protein extract. A number of 31 transcripts were found to be up-regulated. Sequence analyses revealed that they represented genes involved among others in metabolic process, electron transport chain, response to chemical stimulus, and oxidation-reduction processes. Opposite to that 20 down-regulated transcripts were among others related to pseudouridine synthesis, RNA processing, and ribosome biogenesis. The differential expression of three of these transcripts, encoding cytochrome c oxidase subunit 2 (Cox2), NADH-ubiquinone oxidoreductase (ND1), and dyskeratosis congenita 1 protein (DKC1), were confirmed by real-time PCR. The promoted development and the differential gene expression of cultured sporocysts under the influence of head-foot protein extract of native O. hupensis implied not only its ability to improve in vitro culture conditions for intramolluscan stages, it may also represent a priming result with respect to the identification and characterization of factors involved in the parasite-host interplay between S. japonicum and O. hupensis.

Schistosome is the causative agent of human schistosomiasis and related animal disease. Reversible protein phosphorylation plays a key role in signaling processing that are vital for a cell and organism. However, it remains to be undercharacterized in schistosomes. In the present study, we characterized in vivo protein phosphorylation events in different developmental stages (schistosomula and adult worms) of Schistosoma japonicum by using microvolume immobilized metal-ion affinity chromatography (IMAC) pipette tips coupled to nanoLC-ESI-MS/MS. In total, 127 distinct phosphorylation sites were identified in 92 proteins in S. japonicum. A comparison of the phosphopeptides identified between the schistosomula and the adult worms revealed 30 phosphoproteins co-detected in both of the two worms. These proteins included several signal molecules and enzymes such as 14-3-3 protein, cysteine string protein, heat shock protein 90, epidermal growth factor receptor pathway substrate 8, proliferation-associated protein 2G4, peptidyl-prolyl isomerase G, phosphofructokinase and thymidylate kinase. Additionally, the phosphorylation sites were examined for phosphorylation specific motif and evolutionarily conservation. The study represents the first attempt to determine in vivo protein phosphorylation in S. japonicum by using a phosphoproteomic approach. The results by providing an inventory of phosphorylated proteins may facilitate to further understand the mechanisms involved in schistosome development and growth, and then may result in the development of novel vaccine candidates and drug targets for schistosomiasis control., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Schistosomiasis is an important global public health problem, as millions of people are at risk of acquiring this infection. An ideal method for sustainable control of schistosomiasis is using a vaccine alone or in combination with drugs. In the present study, we cloned the SjGALE gene and generated the expression product in E. coli. The expression level of SjGALE during different developmental stages of S. japonicum was evaluated by real-time RT-PCR and western blotting. Immunolocalization indicated that the protein was mainly located on the tegument of the parasite. Infection of rSjGALE-immunized mice demonstrated a 34% and 49% reduction of the mean worm burden and liver egg burden, respectively, in two independent experiments, indicating immune protection. The liver egg count from each female adult worm was significantly reduced by 63% in the two trials. The cytokine profile and IgG isotype analysis demonstrated the induction of a Th1 immune profile in response to immunization with this protein, further suggesting protection against infection. In conclusion, these findings indicated that SjGALE is a potential vaccine against S. japonicum.

Objective: To investigate the changes of sensitivity to praziquantel (PZQ) about PZQ-resistant isolates of Schistosoma japonicum established in laboratory by means of the resistance-inducement method during the stages of adult worms, cercariae and miracidia, so as to provide the basis for establishing the sensitivity-detecting technique to praziquantel., Methods: A Jiangsu laboratory-maintaining isolate and a Hunan field-collecting isolate of S. japonicum that were never treated with PZQ were as PZQ-susceptible isolates, and two PZQ-induced isolates that were established via drug-treated passage in laboratory were as PZQ-resistant isolates. Mice were infected with S. japonicum cercariae collected from above four isolates each. Thirty-five days after the infection, the mice were divided into 6 groups and administered orally with PZQ at dosages of 0, 37.5, 75, 150, 300 mg/kg and 600 mg/kg, respectively. All the mice were sacrificed two weeks after the treatment, and all the adult worms in the hepatic and portomesenteric veins were recovered and counted. The mean worm burden and reductions were calculated and input into Graphpad Prism 5.0 software, and the PZQ ED50 values of four isolates were calculated by the software. The cercariae of above four isolates were exposed to 10(-5), 5 x 10(-6), 10(-6), 5 x 10(-7), 10(-7) mol/L PZQ solutions for 20, 40, 60, 80, 100 min and the changes of tail shedding were observed under a dissecting microscope, then the tail shedding rates of cercariae were calculated. The miracidia of above four isolates were exposed to 5 x 10(-6), 10(-6), 5 x 10(-7), 10(-7) mol/L PZQ solutions for 1, 3 and 5 min and the morphological changes were observed under a dissecting microscope, then the morphological change rates of miracidia were calculated., Results: The PZQ ED50 values of PZQ-susceptible and PZQ-resistant isolates of Jiangsu were 147.7 mg/kg and 565.5 mg/kg, respectively, and the PZQ ED50 values of PZQ-susceptible and PZQ-resistant isolates of Hunan were 151.8 mg/kg and 467.2 mg/kg, respectively. When the cercariae were exposed to 10(-5) mol/L PZQ solution over 20 min, the tail shedding rate of cercariae from PZQ-susceptible isolate of Jiangsu was 68.8%, and the tail shedding rate of cercariae from PZQ-resistant isolate of Jiangsu was 38.2% (P < 0.01). When the cercariae were exposed to 10(-7) mol/L PZQ solution over 100 min, the tail shedding rate of cercariae from PZQ-susceptible isolate of Jiangsu was 15.9%, and the tail shedding rate of cercariae from PZQ-resistant isolate of Jiangsu was 6.7% (P < 0.01). When the cercariae were exposed to 10(-5) mol/L PZQ solution over 20 min, the tail shedding rates of cercariae from PZQ-susceptible isolate of Hunan was 59.4%, and the tail shedding rates of cercariae from PZQ-resistant isolate of Hunan was 54.6% (P < 0.05). When the cercariae were exposed to 5 x 10(-7) mol/L PZQ solution over 40 min, the tail shedding rates of cercariae from PZQ-susceptible isolate of Jiangsu was 34.3%, and the tail shedding rates of cercariae from PZQ-resistant isolate of Jiangsu was 18.4% (P < 0.01). When the miracidia were exposed to 5 x 10(-7) mol/L and 10(-7) mol/L PZQ solutions for 1, 3 and 5 min respectively, the morphological change rates of miracidia from PZQ-susceptible isolates of Jiangsu and Hunan were significantly higher than those of PZQ-resistant isolates (P < 0.01)., Conclusions: PZQ-resistant isolates of S. japonicum has been established in mice with sub-curative doses of PZQ by artificial selection in laboratory, and their sensitivities to PZQ are significantly lower than those of the isolates never treated with PZQ. The drug-resistance could exhibit in the stages of adult worms, cercariae and miracidia. The PZQ ED50 value of adult worms, the tail shedding rates of cercariae and the morphological change rates of miracidia as quantitative indicators can be used for monitoring the S. japonicum sensitivity to PZQ.

Peptides, bound to the tegument of live Schistosoma japonicum schistosomula, were differentially screened by phage display in vitro using three rounds of reverse absorption and bio-panning. Three M13 phage peptides were isolated and identified by determination of their recovery rate, immunohistochemical localization, immunoblot analysis, and their anti-schistosomal effects in vivo and in vitro. Of the three, M13 phage peptide ZL4 (MppZL4, YSGLQDSSLRLR, 1.4kDa, pI 8.8) bound to the tegument of mechanically transformed schistosomula and to other developmental stages of S. japonicum from the mammalian host. By contrast, MppZL4 did not bind to the surface of cercariae. To further examine its binding properties, MppZL4 was conjugated to Rhodamine B (RhB-YSGLQDSSLRLR, RhB-ZL4) and a peptide control (RhB-AIPYFSGILQWR, RhB-12P) was similarly synthesized. The binding capacities of RhB-ZL4 to the surface membrane of S. japonicum schistosomula in vitro and of S. japonicum adult worms in vivo were examined and revealed specificity for binding. When examined for anti-parasite activity, both MppZL4 and RhB-ZL4 exhibited a potent schistosomicidal effect in vitro. Further MppZL4 also affected the growth and development of schistosomula in vivo. These findings extend previous studies showing that phage display techniques can recover polypeptides that bind specifically to living schistosomes and, moreover, that these bound peptides have the potential to inhibit key physiological processes in these parasites. Our findings suggest further that ectogenic polypeptides, which can bind to the tegument of S. japonicum, might be adapted as vectors to deliver experimental probes and/or pharmacologically relevant compounds to the schistosome tegument, including drugs and immunological mediators., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: To investigate the possibility of the emergence of praziquantel resistance in Schistosoma japonicum in Mainland China under drug pressure., Methods: S. japonicum cercaria were released from the infected Oncomelania hupensis snails collected from the marshland in Hunan Province that was endemic for schistosomiasis japonica and raised in the laboratory of Jiangsu Institute of Parasitic Diseases, and mice were infected. O. hupensis snails were infected with miracidia hatched from the schistosome mature eggs that were isolated from the liver of the infected mice. The life cycles of a field isolate and a laboratory passage isolate of S. japonicum were established in laboratory via the cycle of mouse-snail. The mice were infected with 40 cercariae each, 35 days later post-infection, were grouped randomly into control and resistance-induced groups. All the mice in the control group were sacrificed on day 45 post-infection, and any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the worm burdens were calculated. The mice in the resistance-induced group were administered orally with the sub-curative dose of praziquantel, and were sacrificed 22 days post-treatment. Any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the worm burdens and reduction in the worms recovered which were obviously caused by the praziquantel treatment were calculated. The eggs in the liver of the mice in the resistance-induced group were isolated and hatched to yield miracidia, and then the snails were again infected with the newly hatched miracidia to complete the first-passage inducement. After raising in laboratory at 25 degrees C for 60-70 days post-infection, the infected snails were isolated and shed cercaria, and the mice were infected with the newly released cercaria to start a new passage of resistance-inducement. The oral dose of praziquantel for the first-passage inducement was 100 mg/kg, and an additional 100 mg/kg was given every 2-3 passages. The mice were infected with cercariae of the parasite with 8-passge resistance-inducement and the isolate that was not induced, and 35 days post-infection, were administered with praziquantel at a single oral doses of 300 mg/kg and 600 mg/kg respectively. All the mice were sacrificed 14 days post - treatment, and any adult S. japonicum worms in the hepatic and portomesenteric veins were recovered and counted, and the reductions in the worm burdens were calculated to assess the sensitivity of praziquantel in the parasites after 8-passage resistance-inducement., Results: Two isolates of Jiangsu laboratory passage of Jiangsu and field isolate of Hunan were established in the laboratory, and a total 8-passage resistance -inducement was completed. For the laboratory passage isolate, the worm burden reduction was 22.3% post-treatment with 100 mg/kg praziquantel during the first-passage inducement, and 53.7% post-treatment with 300 mg/kg praziquantel during the 8-passage inducement, appearing that the worm burden reduction increased with the increasing dose of praziquantel. For the field-collected isolate, the worm burden reduction was 66.8% post-treatment with 100 mg/kg praziquantel during the first-passage inducement, and 20.6% post-treatment with 300 mg/kg praziquantel during the 8-passage inducement, indicating that the worm burden reduction markedly decreased with the increasing dose of praziquantel. The worm burden reductions were 71.5% and 97.4% respectively for the mice infected with the non-induced laboratory passage isolate, while administered with praziquantel at doses of 300 mg/kg and 600 mg/kg respectively. After 8-passage treatment with sub-curative praziquantel, the corresponding worm burden reductions decreased to 32.6% and 68.1%, respectively. For the field-collected isolate without inducement, the worm burden reductions in the mice were 70.8% and 97.5% respectively post-treatment with praziquantel at doses of 300 mg/kg and 600 mg/ kg respectively, and the corresponding worm burden reductions decreased to 45.7% and 61.9%, respectively after 8-passage treatment. COCLUSIONS: S. japonicum (strain of Mainland China) is able to develop resistance to praziquantel under continuous drug pressure. However, there are variations in the potential of the emergence of resistance due to various susceptibility of praziquantel among different isolates. The successful establishment of praziquantel-resistant strain of S. japonicum (Mainland China) will provide the basis for exploring the mechanism of praziquantel resistance in S. japonicum, and developing related techniques to detect and monitor praziquantel resistance.

Objective: To evaluate the efficiency of rectal administration of praziquantel in the treatment of schistosomiasis in mice., Methods: Forty mice were divided into 4 groups. Each mouse was infected with Schistosoma japonicum cercariae 40 +/- 2. Forty-two days after the infection, the mouse was rectally administered with different doses of praziquantel. In the first, second and third group, each mouse was given 100, 200 mg/kg, and 400 mg/kg single dose of praziquantel, and the fourth group was a blank control group. One week after the administration, all the mice were sacrificed and the worm reduction rate, reduction rate of liver eggs, and matching reduction rate were calculated. RESULTS The worm reduction rate and matching reduction rate were 57.63% and 76.60% respectively in the 200 mg/kg group, and 49.15% and 51.06% respectively in the 400 mg/kg group, which were better than those in the 100 mg/kg group., Conclusion: Rectal administration of praziquantel has good efficiency in the treatment of schistosomiasis in mice; therefore, it provides a new option for the prevention and control of animal schistosomiasis.

Background: Schistosomiasis japonica, caused by contact with Schistosoma japonicum cercaria-infested water when washing, bathing or production, remains a major public-health concern in China. The purpose of the present study was to investigate the effect of a suspension concentrate of niclosamide (SCN) on killing cercaria of S. japonicum that float on the water surface, and its toxicity to fish, so as to establish an emergency-treatment intervention for rapidly killing cercaria and eliminating water infectivity., Results: At 30 min after spraying 100 mg/L SCN, with niclosamide dosages of 0.01, 0.02, 0.03, 0.04 g/m², the water infectivity reduced significantly and no infectivity was found at 60 min after spraying SCN. The surface of static water was sprayed with 100 mg/L SCN, the peak concentration was found at 0 min, and the solution diffused to site with a water depth of 10 cm after 10 min. 30 min later, SCN diffused to the whole water body, and distributed evenly. After spraying 100 mg/L SCN onto the surface of the water with a volume of(3.14 × 202 × 50)cm³, with niclosamide dosages of 0.02 g/m², 96 h later, no death of zebra fish was observed., Conclusions: By spraying 100 mg/L SCN, with a niclosamide dosage of 0.02 g/m² onto the surface of S. japonicum-infested water, infectivity of the water can be eliminated after 30-60 min, and there is no evident toxicity to fish. This cercaria-killing method, as an emergency-treatment intervention for infested water, can be applied in those forecasting and early warning systems for schistosomiasis.

This paper reports the effective treatment of Schistosoma japonicum in a mouse model with long-acting praziquantel (PZQ)-loaded poly(ε-caprolactone) implants. The implants yielded stable, high plasma PZQ concentrations ranging 100-1600 ng/mL during the 40-day investigation period. For assessment of efficacy, the implants were implanted into mice immediately after infection and at 1, 2, 3 and 4 weeks after infection to treat the schistosomes at different developmental stages. All the mice were sacrificed at 6 weeks after infection for worm and egg recovery, worm morphology examination, and histopathological analysis of implantation site tissues. The worm burdens, egg burdens, and numbers of miracidia hatched from the retrieved eggs for all the implant-treated groups (except groups T2-A, T4 and T5) were reduced by 100% when compared with the control group. From groups T2-A, T4 and T5, some schistosome debris was recovered. Eggs were found in only group T5 for which the time between infection and implantation was 4 weeks, which enabled the maturation of juvenile female schistosomes into adult ones that lay eggs. Histopathological observations of implantation tissue showed no evidence of granulomatous foreign-body or lymphoid cell aggregation, demonstrating good biocompatibility of the PZQ implants. These results demonstrate that the long-acting PZQ implants can kill schistosomes at any developmental stages and attenuate/avoid the associated liver damage., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Tetraspanins (TSPs), also known as members of the trans-membrane 4 super-family (TM4SF), comprise an assemblage of surface antigens reported in eukaryotic organisms. In the work presented here, six novel TSP proteins from the human blood fluke Schistosoma japonicum (S. japonicum) were produced and analyzed through a combination of bioinformatics and experimental approaches., Results: Six novel TSP proteins of Schistosoma japonicum (designated as Sj-TSP-#1~6) contained four trans-membrane regions and one large extracellular loop (LEL) with a conserved CCG motif. Size of the proteins varied from 227 to 291 amino acid residues. All the six proteins were produced in E.coli and immune sera to each protein were prepared. Analysis of transcription profiles of the proteins by RT-PCR showed that Sj-TSP-#4 was transcribed only in the egg stage while transcription of the Sj-TSP-#2 was detected in female worms but not in males. The similar results were obtained by Western blot. Immunolocalization of the TSP proteins by immunofluorescence assay showed that the Sj-TSP-#2, Sj-TSP-#5 and Sj-TSP-#6 were located in the tegument of worms., Conclusions: This study provided six novel TSP members of S. japonicum including their sequences and recombinant proteins. Availability of the novel proteins and information on their expression profile and location provided a basis for further investigation of the TSP proteins for their biological functions and as vaccine candidates.

Hemocyanin, a giant oxygen transport protein which is usually found in many arthropods and mollusks was isolated and purified from Oncomelania hupensis. In this study, we showed that Oncomelania hupensis hemocyanin (OhH) shared carbohydrate epitopes with different developmental stages of Schistosoma japonicum (Cercaria, Schistosomulum, Adult worm and Egg) and exhibited serological cross-reaction with these stages of S. japonicum immune sera, which had a potential for use in diagnostic and therapeutic studies of schistosomasis. OhH was used as a vaccine in combination with Freund's adjuvant to evaluate the induction of immune responses and protection against S. japonicum infection in mice. Mice immunized with OhH induced a Th1 type of immune responses. Strong protection against S. japonicum were observed in adult worm and egg burdens after 42 days post-challenge, which showed a significant worm reduction of 52.5% and egg reduction of 69.2% compared to the control groups, respectively. These results indicated that OhH was a potential candidate to compose an anti-schistosome vaccine., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: To study the early immune activation and its dynamic changes between the attenuated cercariae immunized mice and the normal infected mice., Methods: The dendritic cell surface molecules CD11c and T cell surface molecule CD25 expression differences and CD3+CD25+/CD3+ T ratio of the early spleen and/or lung of the attenuated cercariae immunized mice and normal mice were assayed and compared by FCM and IHC, and the immune activation and dynamics of T cells were analyzed., Results: CD3+CD25+CD3+ T ratio in the spleen cells 7 days post-infection in the immunized group and the normal infected group were (19.52 +/- 3.65)% and (22.12 +/- 3.24)%, respectively; the rates of 14 days and 21 days post-infection were (28.73 +/- 3.94)%, (13.68 +/- 3.64)% and(26.43 +/- 0.40)%, (14.42 +/- 2.24)%, respectively. The expressions of CD11c+DC in the lung of the two groups were (1.05 +/- 0.16)%, (0.96 +/- 0.15)%, (1.34 +/- 0.15)%, (1.09 +/- 0.17)%, (1.49 +/- 0.14)%, (0.97 +/- 0.16)%, respectively; the expressions in the spleen were (2.05 +/- 0.26)%, (1.95 +/- 0.18)%, (2.24 +/- 0.25)%, (2.17 +/- 0.25)%, and (2.18 +/- 0.26)%, (2.06 +/- 0.18)%, respectively, on the 7, 14 and 21 days post-infection. The expressions of CD25+T cells in the lung of the two groups were (1.24 +/- 0.13)%, (1.17 +/- 0.16)%, (1.48 +/- 0.11)%, (1.25 +/- 0.13)%, and (1.55 +/- 0.14)%, (0.97 +/- 0.12)%, respectively; the expressions in the spleen were (3.25 +/- 0.22)%, (2.93 +/- 0.20)%, (4.57 +/- 0.23)%, (3.69 +/- 0.24)% and (4.28 +/- 0.24)%, (3.86 +/- 0.26)%, respectively, on the 7, 14 and 21 days post-infection. The CD3+CD25+/CD3+T rate in the infection control group was significantly higher than that in the cercariae attenuated group, while 14, 21 days post-infection the rates of the attenuated group were significantly higher than those in the normal control group. On the 7, 14 and 21 days post-infection, the lung tissue of the attenuated cercariae immunized mice raised more CD11c+ DC and CD25+ T cells than that of the normal infected mice did., Conclusions: The activation of T cells of the immune group and the activation of pulmonary dendritic cells are higher than those in the control group 7 and 14 days post-infection, suggesting that attenuated cercariae in the lungs can raise more antigen presenting cells and their activation.

Background: The gene regulation mechanism along the life cycle of the genus Schistosoma is complex. Small non-coding RNAs (sncRNAs) are essential post transcriptional gene regulation elements that affect gene expression and mRNA stability. Preliminary studies indicated that sncRNAs in schistosomal parasites are generated through different pathways, which are developmentally regulated. However, the data of sncRNAs of schistosomal parasites are still fragmental and a complete expression profile of sncRNAs during the parasite development requires a deep investigation., Methodology/principal Findings: We employed high-throughput genome-wide transcriptome analytic techniques to explore the dynamic expression of microRNAs (miRNAs) and endogenous siRNAs (endo-siRNAs) of Schistosoma japonicum covering the free-living cercarial stage and all stages in the definitive host. This led us to analyze over 70 million clean reads represented both high and low abundance of the small RNA population. Patterns of differential expression of miRNAs and endo-siRNAs were observed. MiRNAs was twice more than endo-siRNAs in cercariae, but gradually decreased along with the development of the parasite. Both small RNA types were presented in equal aboudance in lung-stage schistosomula, while endo-siRNAs accumulated to 6 times more than miRNAs in adult female worms and hepatic eggs. Further, miRNAs were found mainly derived from genes located in the intergenic regions, while endo-siRNAs were mainly generated from transposable elements (TEs). The expression pattern of TE-siRNAs, as well as the pseudogene-derived siRNAs clustered in mRNAs of cytoskeletal proteins, stress proteins, enzymes related to energy metabolism also revealed distinction throughout different developmental stages. Natural antisense transcripts (NATs)-related siRNAs accounted for minor proportion of the endo-siRNAs which were dominantly expressed in cercariae., Conclusions/significance: Our results represented a comprehensive expression profile of sncRNAs in various developmental stages of S. japonicum with high accuracy and coverage. The data would facilitate a deep understanding of the parasite biology and potential discovery of novel targets for the design of anti-parasite drugs.

Objective: To understand the susceptibilities of Oncomelania hupensis snails to Schistosoma japonicum miracidia from different hosts., Methods: The Schistosoma japonicum eggs from different hosts, such as rabbits, cattle and mice were collected. These eggs were incubated for miracidia, respectively. Each snail from the same site was exposed to 5 miracidia of Schistosoma japonicum from different hosts. The infected snails were fed in the laboratory for two months. Then all the snails were dissected and observed under the dissecting microscope in order to know the infection rate of snails., Results: In the experiment group, the infection rates of snails infected with miracidia from rabbits, cattle and mice were 1.42%, 8.67% and 19.87%, respectively, the mortality rates were 29.5%, 13.5% and 24.5%, respectively. However, the infection rates of snails in the control group were 2.63%, 2.02% and 11.66%, respectively, and the mortality rates were 24.0%, 49.5% and 18.5%, respectively., Conclusion: The susceptibilities of Oncomelania snails to Schistosoma japonicum miracidia from 3 kinds of hosts are significantly different.

Objective: To clone and express Schistosoma japonicum Toll like receptor interacting protein (SjTollip) in prokaryotic expression system and analyze its stage-specific transcription and expression., Methods: The encoding sequence selected from S. japonicum cDNA library was amplified by PCR. The SjTollip gene obtained was subcloned into pET-28a, then transformed into E.coli BL21 and induced with IPTG for expression. The expressed protein was purified with Ni-NTA resin. Total RNA were extracted from different stages of S. japonicum. The immune rabbit sera were prepared by immunizing New Zealand white rabbits with purified recombinant SjTollip protein. RT-PCR and Western blotting were used to analyze the transcription and expression level at the different stages and the immunogenicity., Results: The expression vector of SjTollip/pET-28a was constructed and expressed as inclusion bodies (Mr 24 000). The recombinant protein rSjTollip was specifically recognized by the S. japonicum-infected rabbit serum. SjTollip showed lower transcription level in stages including cercariae and male worms. Western blotting analysis showed that the target protein was detected in all stages. The expression level in stages including schistosomulum and female worm was much higher., Conclusions: The SjTollip transcription and expression level at the different stages of S. japonicum is different, and this gene might be a potential candidate for target of vaccine, drug and diagnosis.

Objective: To observe the destructive effect of eggs of Schistosomajaponicum on the splenic structure in C57BL/6 mice., Methods: The C57BL/6 mice were injected intravenously with eggs of S. japonicum or soluble egg antigen (SEA), or the eggs were injected surgically into the spleens of the mice. Four weeks later, the mice were sacrificed, the splenic paraffin sections were stained with H & E and the splenic structures were observed. Other two groups of mice were infected with single-sex and both sexes of schistosome cercariae, respectively. Nine weeks after the infection, the mice were sacrificed, and the splenic structures were observed and compared among the groups aforementioned., Results: The splenic structure of mice injected intravenously with eggs were destroyed, and characterized with decreased number of lymphoid follicles and blurred marginal zone. Lymphoid follicles around the eggs injected into the spleen were also seriously depauperated. The splenic weight (0.15 +/- 0.01) g of the mice infected with single-sex cercariae significantly less than that (0.41 +/- 0.03) g of the mice infected with both sexes cercariae (P < 0.01). However, the splenic structure of the mice infected with single-sex cercariae kept integrated., Conclusion: Eggs of S. japonicum have a destructive effect on the splenic structure in C57BL/6 mice.

Over the past years there has been an increasing trend in the application of mathematical models in the studies and control of infectious disease. With development of computing technologies and increasing access to them, it becomes appealing for public health professionals to use mathematical models in their work. In this paper based on our previous experience working on environmental determinants and control of schistosomiasis in the hilly and mountainous regions of Sichuan Province, we introduce principles and basic procedures in building a mathematical model for schistosomiasis transmission; we then illustrate the use of a mathematical model to integrate data from diverse sources and to aid in designing control strategies, assessing progress, and evaluating surveillance programs.

Objective: To develop an aetiology detection method with higher sensitivity and accuracy for schistosomiasis japonica., Methods: The fresh fecal samples were collected from residents of a schistosomiasis japonica endemic village in Laozi County of Hubei Province. Two samples per person were collected in two consecutive days. Each sample was examined three times with Kato-Katz technique, then the negative cases were re-examined by the stool hatching test after the egg concentration with nylon-tissue bags. The detective rates and the relation with egg per gram (EPG) of different methods were analyzed and compared. The epidemiology data were collected as well., Results: A total of 562 residents were examined by Kato-Katz technique, and 67 of them were positive. The samples of 495 negative persons were re-examined by using the hatching test after the egg concentration with nylon-tissue bags. Six persons were positive. The total number of positive cases was 73. The rates of Kato-Katz technique with three slides of one sample and six slides of two samples were 65.8% and 91.8%, respectively. The detective rate was increased with the increase of examining slides. The geometrical averages of EPG from one slide to six slides were 61.12, 31.92, 23.71, 20.88, 19.35 and 8.83, respectively. The accuracy of EPG increased with the increase of numbers of slides., Conclusion: Kato-Katz technique with six slides of two samples is applicable in diagnosis of schistosomiasis japonica in order to achieve higher detective rate and more accurate EPG.