Development of a Gaussia luciferase immunoprecipitation assay for detecting Schistosoma japonicum infection.

Timely and accurate diagnosis of Schistosoma infection is important to adopt effective strategies for schistosomiasis control. Previously, we demonstrated that Schistosoma japonicum can secret extracellular vesicles and their cargos may serve as a novel type of biomarkers for diagnosing schistosomiasis. Here, we developed a Gaussia luciferase immunoprecipitation assay combined with S. japonicum extracellular vesicle (SjEV) protein to evaluate its potential for diagnosing schistosomiasis. A saposin-like protein (SjSLP) identified from SjEVs was fused to the Gaussia luciferase as the diagnostic antigen. The developed method showed good capability for detecting S. japonicum infection in mice and human patients. We also observed that the method could detect Schistosoma infection in mice as early as 7 days of post-infection, which showed better sensitivity than that of indirect ELISA method. Overall, the developed method showed a good potential for detecting Schistosoma infection particularly for early stage, which may provide an alternative strategy for identify Schistosoma infection for disease control.

Identification of Schistosoma japonicum GSK3ß interacting partners by yeast two-hybrid screening and its role in parasite survival.

Schistosoma is the causative agent of schistosomiasis, a common infectious disease distributed worldwide. Our previous phosphoproteomic analysis suggested that glycogen synthase kinase 3 (GSK3), a conserved protein kinase in eukaryotes, is likely involved in protein phosphorylation of Schistosoma japonicum. Here, we aimed to identify the interacting partners of S. japonicum GSK3ß (SjGSK3ß) and to evaluate its role in parasite survival. Toward these ends, we determined the transcription levels of SjGSK3ß at different developmental stages and identified its interacting partners of SjGSK3ß by screening a yeast two-hybrid S. japonicum cDNA library. We further used RNA interference (RNAi) to inhibit the expression of SjGSK3ß in adult worms in vitro and examined the resultant changes in transcription of its putative interacting proteins and in worm viability compared with those of control worms. Reverse transcription-quantitative polymerase chain analysis indicated that SjGSK3ß is expressed throughout the life cycle of S. japonicum, with higher expression levels detected in the eggs and relatively higher expression level found in male worms than in female worms. By screening the yeast two-hybrid library, eight proteins were identified as potentially interacting with SjGSK3ß including cell division cycle 37 homolog (Cdc37), 14-3-3 protein, tegument antigen (I(H)A), V-ATPase proteolipid subunit, myosin alkali light chain 1, and three proteins without recognized functional domains. In addition, SjGSK3ß RNAi reduced the SjGSK3ß gene transcript level, leading to a significant decrease in kinase activity, cell viability, and worm survival. Collectively, these findings suggested that SjGSK3ß may interact with its partner proteins to influence worm survival by regulating kinase activity.

Roles of microRNAs in T cell immunity: Implications for strategy development against infectious diseases.

T cell immunity plays a vital role in pathogen infections. MicroRNA (miRNAs) are small, single-stranded noncoding RNAs that regulate T cell immunity by targeting key transcriptional factors, signaling proteins, and cytokines associated with T cell activation, differentiation, and function. The dysregulation of miRNA expression in T cells may lead to specific immune responses and can provide new therapeutic opportunities against various infectious diseases. Here, we summarize recent studies that focus on the roles of miRNAs in T cell immunity and highlight miRNA functions in prevalent infectious diseases. Additionally, we also provide insights into the functions of extracellular vesicle miRNAs and attempt to delineate the mechanism of miRNA sorting into extracellular vesicles and their immunomodulatory functions. Moreover, methodologies and strategies for miRNA delivery against infectious diseases are summarized. Finally, potential strategies for miRNA-based therapies are proposed.

In silico analysis of endogenous siRNAs associated transposable elements and NATs in Schistosoma japonicum reveals their putative roles during reproductive development.

Schistosomiasis is a neglected tropical disease caused by trematode of the genus Schistosoma. Successful reproductive development is critical for the production of eggs, which are responsible for host pathology and disease dissemination. Endogenous small non-coding RNAs play important roles in many biological processes such as protection against foreign pathogens, cell differentiation, and chromosomal stability by regulating target gene expression at the transcriptional and post-transcriptional levels. In this study, we performed in silico analysis of endogenous small non-coding RNAs in different stages, and sex of S. japonicum focusing on endogenous small interfering RNAs (endo-siRNAs) generated from transposable elements (TEs) and natural antisense transcripts (NATs). Both total and unique siRNA populations show 18-30 nt in length, but the predominant size was 20 nt and the leading first base was adenosine. Sense TE-derived endo-siRNAs reads were higher than antisense reads at different relative positions of TEs, whereas no such difference was observed for NAT-derived endo-siRNAs. TE- and NAT-derived endo-siRNAs were more enriched in the male compared to female worms, with the higher relative expression in early phase of pairing. Putative targets of endo-siRNAs indicated more of them in males (106 and 66) than in females (6 and 23) for TE- and NAT-derived endo-siRNAs, respectively. Our preliminary study revealed vital role of endo-siRNAs during the reproductive development of S. japonicum and provide clues for putative novel targets to suppress worm reproduction and direction for effective anti-schistosomal drug development.

Molecular characterization, expression profile, and preliminary evaluation of diagnostic potential of CD63 in Schistosoma japonicum.

Schistosomes are the causative agents of human schistosomiasis, which is endemic in tropical and subtropical zones. CD63 is a member of the tetraspanin protein family widely expressed among eukaryotes. Previously, we identified a CD63 homolog from extracellular vesicles isolated from Schistosoma japonicum. In this study, we characterized this CD63 homolog using a molecular approach and evaluated the potential of its recombinant protein for the diagnosis of schistosomiasis. A sequence alignment indicated that S. japonicum CD63 (SjCD63) has sequence identities of 76 and 28% with S. mansoni and human CD63, respectively. A phylogenetic analysis displayed that S. japonicum CD63 is related to S. mansoni and Opisthorchis viverrini CD63. The cDNA of SjCD63 was 740 bp long with an expected protein size of 23.58 kDa. A RT-qPCR analysis revealed significantly higher expression of SjCD63 mRNA in adult worms on days 21, 28, and 35 than in 7-day schistosomula, cercariae, and eggs. In addition, recombinant SjCD63 protein detected by ELISA revealed significantly higher optical density values compared to that of the negative control in both S. japonicum-infected mouse and rabbit sera, providing preliminary evidence for its diagnostic potential. Overall, these results provide insight into the molecular properties of SjCD63, its expression profiles, and its preliminary diagnostic potential.

Molecular characterization and expression profile of nanos in Schistosoma japonicum and its influence on the expression several mammalian stem cell factors.

Pluripotent stem cells, called neoblasts, are well known for the regenerative capability and developmental plasticity in flatworms. Impressive advancement has been made in free-living flatworms, while in case of its parasitic counterpart, neoblast-like stem cells have attracted recent attention for its self-renewal and differentiation capacity. Nanos is a key conserved post-transcriptional regulator critical for the formation, development, and/or maintenance of the pluripotent germ line stem cell systems in many metazoans including schistosomes. In the present study, we report the molecular cloning and expression of nanos orthologous genes nanos in Schistosoma japonicum (Sjnanos). The cDNA of Sjnanos is 826 bp long, containing an open reading frame (ORF) for 223 amino acid long protein. qRT-PCR analysis shown that Sjnanos was differently expressed in several stages of schistosomes with relatively high level in schistosomula. Additionally, Sjnanos was expressed highly in adult females compared to adult males. Transfection of recombinant plasmid for expressing Sjnanos resulted in significant proliferation and increased expression of several stem cell factors in mammalian cells. Overall, our preliminary study provides the molecular basis to further functionally characterize Sjnanos in S. japonicum.

Cysticercus fasciolaris infection induced oxidative stress and apoptosis in rat liver: a strategy for host-parasite cross talk.

Parasitic helminths have developed various strategies to induce or inhibit apoptosis in the cells of their host, thereby modulating the host's immune response and aiding dissemination to the host. Cysticercus fasciolaris, the larval form of Taenia taeniaeformis, parasitized different intermediate hosts like rats, rabbits, etc. and is cosmopolitan in distribution. In the present study, we have investigated host-parasite interactions and the resulting effect of C. fasciolaris in the liver of rat. Histology of the infected livers showed dilation and damages of hepatic cells near the parasite. Infected liver cells showed an increase in DNA fragmentation and chromatin condensation compared to the normal liver. Acridine orange and ethidium bromide dual staining revealed the presence of apoptotic cells in the infected liver. The decline in the mitochondrial membrane potential in the infected liver suggested that the observed apoptosis is mitochondria mediated. Occurrence of an elevated level of active executioner caspases 3/7 in the infected rat liver further confirms the occurrence of apoptosis. Different antioxidant enzymes were also evaluated and revealed a notable decline in the level of glutathione and glutathione-S-transferase activity leading to the augmented generation of reactive oxygen species. Results of the present study revealed that C. fasciolaris infection leads to apoptosis in the liver of rats which may be a surviving strategy for the parasitic larvae.

Resveratrol- and α-viniferin-induced alterations of acetylcholinesterase and nitric oxide synthase in Raillietina echinobothrida.

Phytostilbenes, like resveratrol and α-viniferin, which occur mainly in the plants and belong to the families Cyperaceae, Vitaceae, and Gnetaceae are extensively popular for their medicinal and nutritional properties. In Northeast India, the Jaintia tribes consume these phytochemicals through aqueous extract of the medicinal plant Carex baccans to control helminthiasis. The present study aimed to investigate the inhibitory effect of the phytochemicals on neurotransmitters and its related enzymes in helminth parasite Raillietina echinobothrida. Viability of the parasites exposed to the phytostilbenes and extent of inhibition of cholinergic and nitrergic enzymes were evaluated in comparison to reference anthelmintic drug praziquantel and two known enzyme inhibitors, namely Nω-nitro-L-arginine and pyridostigmine. On exposure to resveratrol, α-viniferin, and reference drug praziquantel, the parasites ceased movement at 9.37, 11.38, and 0.24 h followed by death at 23.65, 34.13, and 1.87 h, respectively. Exposed parasites also showed a significant decrease in the activity of acetylcholinesterase (46.101, 65.935, and 63.645%) and nitric oxide synthase (61.241, 55.046, and 29.618%) in comparison to the controls. In addition, a decreased trend in nitric oxide (NO) level was also detected in the tissue of different phytochemical-exposed parasites compared to control. The present study suggests that anthelmintic potential of both the phytochemicals is mediated through inhibition of two vital enzymes which play diverse role in intracellular communications through neuromuscular system.

In vivo anthelmintic activity of Carex baccans and its active principle resveratrol against Hymenolepis diminuta.

Anthelmintic resistance against most of the commercial drugs is a great threat to humans as well as the veterinary live stocks. Hence, new treatment strategies to control helminth infections are essential at this hour. Carex baccans Nees has been traditionally used by Jaintia tribes in Northeast India to get rid of intestinal worm infections. Therefore, the present study was conducted to evaluate in vivo cestocidal activity of root tuber extract of C. baccans and its active component resveratrol against the zoonotic cestode Hymenolepis diminuta in the experimental model rat. The cestocidal activity was determined by monitoring the eggs per gram (EPG) counts in faeces of different treated groups. The result showed that the highest dose of the plant extract (50 mg/kg) and resveratrol (4.564 mg/kg body weight) has significant anthelmintic efficacy against H. diminuta. Crude extract of the plant as well as resveratrol reduced EPG count (56.012 and 46.049 %) and also resulted in decreased worm burden by 44.287 and 31.034 %, respectively. The efficacy of the crude extract and resveratrol can be compared to the reference drug praziquantel. The results exhibits considerable cestocidal potential of root tuber crude extract of C. baccans and resveratrol and justify its folklore use.

α-Viniferin-Induced Structural and Functional Alterations in Raillietina echinobothrida, a Poultry Tapeworm.

α-Viniferin, an active component of the plant Carex baccans L., is known for its anticancer, antidiabetic, and anti-inflammatory properties. In Northeast India, different tribes traditionally consume C. baccans to control intestinal helminth infections. Therefore, the present study was carried out to assess the extent of tegumental alteration caused by α-viniferin in Raillietina echinobothrida, a widely prevalent poultry helminth in northeast India. Helminths were exposed in vitro to various doses of α-viniferin (50, 100, and 200 µM/mL of physiological buffered saline) and their motility and mortality were recorded. Stereoscan observations on the parasite exposed to the active compound showed extensive distortion and destruction of the surface fine topography of the tegument compared with controls. The compound also caused extensive damage to the tegument by disintegration of microtriches, disorganization of muscle bundles, and loss of cellular organelles combined with distortion and disruption of the plasma membrane, nuclear membrane, nucleolus, mitochondrial membrane, and cristae. Histochemical and biochemical studies carried out parasites exposed to α-viniferin revealed a decline in the activity of vital tegumental enzymes like acid phosphatase, alkaline phosphatase, and adenosine triphosphatase. Extensive structural and functional alterations observed in the treated parasites are indicative of efficient cestocidal activity of the compound.

Exogenous modification of EL-4 T cell extracellular vesicles with miR-155 induce macrophage into M1-type polarization.

Extracellular vesicles (EVs) show promising potential to be used as therapeutics, disease biomarkers, and drug delivery vehicles. We aimed to modify EVs with miR-155 to modulate macrophage immune response that can be potentially used against infectious diseases. Primarily, we characterized T cells (EL-4) EVs by several standardized techniques and confirmed that the EVs could be used for experimental approaches. The bioactivities of the isolated EVs were confirmed by the uptake assessment, and the results showed that target cells can successfully uptake EVs. To standardize the loading protocol by electroporation for effective biological functionality, we chose fluorescently labelled miR-155 mimics because of its important roles in the immune regulations to upload them into EVs. The loading procedure showed that the dosage of 1 µg of miRNA mimics can be efficiently loaded to the EVs at 100 V, further confirmed by flow cytometry. The functional assay by incubating these modified EVs (mEVs) with in vitro cultured cells led to an increased abundance of miR-155 and decreased the expressions of its target genes such as TSHZ3, Jarid2, ZFP652, and WWC1. Further evaluation indicated that these mEVs induced M1-type macrophage polarization with increased TNF-α, IL-6, IL-1ß, and iNOS expression. The bioavailability analysis revealed that mEVs could be detected in tissues of the livers. Overall, our study demonstrated that EVs can be engineered with miR-155 of interest to modulate the immune response that may have implications against infectious diseases.

Schistosoma japonicum extracellular vesicle proteins serve as effective biomarkers for diagnosing parasite infection.

Schistosoma species are the causative agent of schistosomiasis and shows worldwide distribution. There is a great need to develop a sensitive diagnostic approach for controlling the disease. Previously, we identified large numbers of Extracellular Vesicle (EV) proteins from Schistosoma japonicum (S. japonicum), but rarely these proteins have been evaluated for their diagnostic potential. In the present study, we performed bioinformatic analyses of S. japonicum identified EV-associated proteins from the previous study and then identified Schistosoma-specific proteins with potentially secreted capability. Among them, we selected SJCHGC02838 protein, SJCHGC05593 protein, SJCHGC05668 protein and a hypothetical protein (SJHYP) to evaluate their diagnostic potential for detecting S. japonicum infection. First, we determined the expression of these four proteins at the transcript levels using qRT-PCR and revealed that all these genes showed higher expression in adult stage. Then, we cloned the full-length cDNA for each protein into a prokaryotic expression vector and successfully generated the recombinant proteins. Upon the purification of recombinant proteins, we developed an indirect ELISA method to evaluate the diagnostic potential of these purified recombinant proteins. The results showed high sensitivity for detecting Schistosoma infection. Additionally, these proteins also displayed a good potential for detecting Schistosoma infection, especially SJCHGC05668 protein at an early stage. The diagnostic potentials of these recombinant proteins were further evaluated by Western blot and comparatively analyzed by our previously developed cfDNA methods.

Evidence of apoptosis in Raillietina echinobothrida induced by methanolic extracts of three traditional medicinal plants of Northeast India.

The therapeutic benefits of medicinal plants in terms of anthelmintic properties are known since time immemorial in India, particularly among natives of the Northeast India. However, only sporadic and scarce reports on scientific validation of these plants are available. The present study was conducted on the cestode Raillietina echinobothrida, to establish whether the anthelmintic activity of Potentilla fulgens, Alpinia nigra and Millettia pachycarpa was mediated by apoptosis or not. Light microscopic observation following MTT assay revealed the highest percentage of inhibition of viability among the worms by methanol extract of M. pachycarpa (89.33%), followed by A. nigra (65%) and P. fulgens (37%). Ultrastructural observations revealed swelling of mitochondria, disruption of mitochondrial membrane, vacuolization of mitochondria, appearance of apoptotic bodies in the cytoplasm, disintegration of nuclear membrane and nucleolus were very common throughout the tegument. DAPI stained specimens showed typical morphology of apoptosis, like nuclear condensation and fragmentation in the extracts treated parasites. A decrease in mitochondrial membrane potential was also recorded in the treated groups. Confirmatory TUNEL assay and DNA fragmentation assay of the extracts treated parasites also confirmed the apoptotic nature of cell death and is concluded to be responsible for paralysis and death of the parasite.

Metagenomic sequencing for identifying pathogen-specific circulating DNAs and development of diagnostic methods for schistosomiasis.

Timely diagnosis of Schistosoma infection, particularly in the early stage is crucial for identifying infected hosts and then taking effective control strategies. Here, metagenomic next-generation sequencing was used to identify pathogen-specific circulating DNAs (cDNAs) in the sera/plasma of New Zealand rabbits infected with S. japonicum, and the identified cDNAs were validated by PCR and qPCR. Loop-mediated isothermal amplification (LAMP)-based CRISPR-Cas12a and recombinase polymerase amplification-based lateral flow strip (RPA-LF) methods combined with the newly identified cDNA were developed to evaluate the potentials for diagnosing murine and human schistosomiasis. The results indicated that twenty-two cDNAs were identified. The developed LAMP-based CRISPR/Cas12a and RPA-LF methods showed a good potential for diagnosing murine or human schistosomiasis as early as 5 days of post-infection with 5 cercariae infection. In a word, S. japonicum specific cDNAs in circulation of infected hosts could be effective biomarkers for detecting Schistosoma infection particularly for early stages.

Ultrastructural and biochemical alterations in rats exposed to crude extract of Carex baccans and Potentilla fulgens.

The use of plants as a source of medicine is an important component of the health care system in rural India. Carex baccans (Cyperaceae) and Potentilla fulgens (Rosaceae) have been known since ancient times in northeast India for their antitumor, antidiabetic, and antihelmintic properties. The present study was designed to determine the subacute toxicity profile of the root tuber extract of C. baccans and root-peel extract of P. fulgens in Wistar rats. The subacute oral toxicity was conducted using sublethal doses of 40, 50, 100, 150, 200, and 400 mgkg-1 body weights. Surface topographical and ultrastructural observations of liver and intestinal microvilli showed remarkable deformation and disruption, accompanied by quantitative changes in the liver enzymes, i.e., aspartate aminotransferase and alanine aminotransferase in comparison to those of the control group. Apoptotic cell death was observed in the liver cells of rats exposed to both of the plant extracts. A significant increase in splenic lymphocyte count was also observed in rats exposed to the highest concentration of both extracts. The results showed that consumption of the plant extracts at higher doses may cause toxicological effect if treatment continues for a long time.

Genome-wide identification of circular RNAs in adult Schistosoma japonicum.

Circular RNAs (circRNAs) are a class of novel, widespread, covalently closed RNAs that have played an essential role in animal gene regulation. To systematically explore circRNAs in the blood fluke Schistosoma japonicum, we performed RNA sequencing and bioinformatics analysis, and found that hundreds of circRNAs showed gender-associated expression. Among these identified circRNAs, more than 77.54% and 74.73% were putatively derived from the exon region of the genome and some circRNAs showed gender-associated expressions. The functional prediction of circRNAs (circ_003826 and circ_004690) showed potential binding sites and possibly acted as the sponge to regulate microRNAs (miRNAs) sja-miR-1, sja-miR-133 and sja-miR-3504. Altogether, these findings demonstrated that S. japonicum also contains circRNAs, which may have potential regulatory roles during schistosome development.

Characterization of MicroRNA Cargo of Extracellular Vesicles Isolated From the Plasma of Schistosoma japonicum-Infected Mice.

Schistosoma is a genus of parasitic trematodes that undergoes complex migration in final hosts, finally developing into adult worms, which are responsible for egg production and disease dissemination. Recent studies documented the importance of extracellular vesicles (EVs) in the regulation of host-parasite interactions. Herein, we investigated the microRNA (miRNA) profiles of EVs isolated from host plasma at different stages of Schistosoma japonicum infection (lung stage: 3 days post-infection (dpi), and liver stages: 14 and 21 dpi) to identify miRNA cargo potentially involved in the pathogenesis and immune regulation of schistosomiasis. Characterization of the isolated plasma EVs revealed their diameter to be approximately 100 nm, containing typical EV markers such as Hsp70 and Tsg101. Deep sequencing analysis indicated the presence of 811 known and 15 novel miRNAs with an increasing number of differential miRNAs from the lung stage (27 miRNAs) to the liver stages (58 and 96 miRNAs at 14 and 21 dpi, respectively) in the plasma EVs of infected mice compared to EVs isolated from the uninfected control. In total, 324 plasma EV miRNAs were shown to be co-detected among different stages of infection and the validation of selected miRNAs showed trends of abundance similar to deep sequencing analysis. For example, miR-1a-3p and miR-122-5p showed higher abundance, whereas miR-150-3p and miR-126a showed lower abundance in the plasma EVs of infected mice at 3, 14, and 21 dpi as compared to those of uninfected mice. In addition, bioinformatic analysis combined with PCR validation of the miRNA targets, particularly those associated with the immune system and parasitic infectious disease, indicated a significant increase in the expression of Gbp7and Ccr5 in contrast to the decreased expression of Fermt3, Akt1, and IL-12a. Our results suggested that the abundance of miRNA cargo of the host plasma EVs was related to the stages of Schistosoma japonicum infection. Further studies on the roles of these miRNAs may reveal the regulatory mechanism of the host-parasite interaction. Moreover, the differentially abundant miRNA cargo in host EVs associated with S. japonicum infection may also provide valuable clues for identifying novel biomarkers for schistosomiasis diagnosis.

Dynamic miRNA profile of host T cells during early hepatic stages of Schistosoma japonicum infection.

Schistosomes undergo complicated migration in final hosts during infection, associated with differential immune responses. It has been shown that CD4+ T cells play critical roles in response to Schistosoma infections and accumulated documents have indicated that miRNAs tightly regulate T cell activity. However, miRNA profiles in host T cells associated with Schistosoma infection remain poorly characterized. Therefore, we undertook the study and systematically characterized T cell miRNA profiles from the livers and blood of S. japonicum infected C57BL/6J mice at 14- and 21-days post-infection. We observed 508 and 504 miRNAs, in which 264 miRNAs were co-detected in T cells isolated from blood and livers, respectively. The comparative analysis of T cell miRNAs from uninfected and infected C57BL/6J mice blood showed that miR-486b-5p/3p expression was significantly downregulated and linked to various T cell immune responses and miR-375-5p was highly upregulated, associated with Wnt signaling and pluripotency, Delta notch signaling pathways, etc. Whereas hepatic T cells showed miR-466b-3p, miR-486b-3p, miR-1969, and miR-375 were differentially expressed compared to the uninfected control. The different expressions of some miRNAs were further corroborated in isolated T cells from mice and in vitro cultured EL-4 cells treated with S. japonicum worm antigens by RT-qPCR and similar results were found. In addition, bioinformatics analysis combined with RT-qPCR validation of selected targets associated with the immune system and parasite-caused infectious disease showed a significant increase in the expression of Ctla4, Atg5, Hgf, Vcl and Arpc4 and a decreased expression of Fermt3, Pik3r1, Myd88, Nfkbie, Ppp1r12a, Ppp3r1, Nfyb, Atg12, Ube2n, Tyrobp, Cxcr4 and Tollip. Overall, these results unveil the comprehensive repertoire of T cell miRNAs during S. japonicum infection, suggesting that the circulatory (blood) and liver systems have distinct miRNAs landscapes that may be important for regulating T cell immune response. Altogether, our findings indicated a dynamic expression pattern of T cell miRNAs during the hepatic stages of S. japonicum infection.

Transcriptional profiles of genes potentially involved in extracellular vesicle biogenesis in Schistosoma japonicum.

Schistosomiasis is a severe chronic disease caused by parasitic worms of the genus Schistosoma. Recent studies indicate that schistosomes can secrete extracellular vesicles (EVs), which play important regulatory roles in many biological processes. However, the mechanisms underlying EV biogenesis in schistosomes are poorly understood. In this study, we performed bioinformatic analyses and identified several genes putatively involved in EV biogenesis in Schistosoma japonicum, which were then confirmed by PCR. Quantitative transcriptional profiles of the selected genes indicated that they were differentially expressed in male and female worms as well as in the different developmental stages of S. japonicum. Thus, the highest expression of VAMP3 was detected in cercariae, whereas that of ARF6 was detected in eggs. RAB11A and the Syntenin-encoding gene SDCBP were highly expressed in 14-day schistosomula and VPS4A and RAB27A were highly expressed in 35-day-old adult schistosomes. The expression of RAB11A, CHMP4C, VPS4A, and SDCBP was higher in male worms, whereas that of ARF6, VAMP3, and RAB27A was higher in female worms. Our results are expected to provide important clues for understanding the role of EV biogenesis in S. japonicum development.

Detection of circulating cell-free DNA to diagnose Schistosoma japonicum infection.

Schistosomiasis occurs in 240 million people worldwide and is a major public health concern. Thus, early diagnosis and monitoring of schistosomiasis progression are needed to treat patients. Cell-free DNA (cfDNA) is present as fragments of parasite-derived DNA in host body fluids. Detection of this cfDNA in host blood may be a promising diagnostic marker of schistosomiasis. Therefore, in this study, we investigated the potential of internal transcribed spacer 2 (ITS2), a molecular taxonomy and barcoding marker, in diagnosing schistosomiasis using infected rabbit and mice sera. A 192 bp fragment of ITS2 was detected in the serum-isolated DNA from the infected host on different days after infection. We also determined the sensitivity of detecting ITS2 in mice with varying numbers of cercaria: cfDNA was present even in mice with low abundance of the parasite. Overall, our results show that cfDNA may be a potential tool for the early diagnosis and therapeutic evaluation of S. japonicum infection.