Changing seroprevalence of schistosomiasis japonica in China from 1982 to 2020: A systematic review and spatial analysis.

BACKGROUND: Schistosomiasis is a global public health issue. In China, while the seroprevalence of Schistosomiasis japonica has currently reduced to a relatively low level, risk of infection still exists in certain areas. However, there has been a lack of comprehensive research on the long-term trends of national seroprevalence, changes across age groups, and characteristics in spatial distribution, which is crucial for effectively targeting interventions and achieving the goal of eliminating schistosomiasis by 2030. Our study aimed to address this gap by analyzing the long-term trends of Schistosomiasis japonica seroprevalence in China from 1982 to 2020 based on the data from diverse sources spanning a period of 39 years. METHODOLOGY: Seroprevalence data were collected from literature databases and national schistosomiasis surveillance system. Meta-analysis was conducted to estimate the seroprevalence. Joinpoint model was used to identify changing trend and inflection point. Inverse distance weighted interpolation was used to determine the spatial distribution of seroprevalence. PRINCIPAL FINDINGS: The seroprevalence decreased from 34.8% in 1982 to 2.4% in 2020 in China. Before 2006, the seroprevalence was higher in the middle age group, and a pattern of increasing with age was observed afterwards. The areas with high seroprevalence existed in Dongting Lake, Poyang Lake, Jianghan Plain, the Anhui branch of the Yangtze River and some localized mountainous regions in Sichuan and Yunnan provinces. CONCLUSIONS/SIGNIFICANCE: There was a significant decline in the seroprevalence of Schistosomiasis japonica from 1982 to 2020 in China. Nevertheless, schistosomiasis has not been eradicated; thus, implementing precise and personalized monitoring measures is crucial for the elimination of schistosomiasis, especially in endemic areas and with a particular focus on the elderly.

Proliferation of MDSCs may indicate a lower CD4+ T cell immune response in schistosomiasis japonica.

BACKGROUND: Schistosoma japonicum (S. japonicum) is the main species of Schistosoma prevalent in China. Myeloid-derived suppressor cells (MDSCs) are important immunoregulatory cells and generally expand in parasite infection, but there is little research relating to MDSCs in Schistosoma infection. METHODS: Fifty-six S. japonicum-infected patients were included in this study. MDSCs and percentages and absolute cell numbers of lymphocyte subsets, including CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells and natural killer (NK) cells were detected using flow cytometry. The degree of liver fibrosis was determined using color Doppler ultrasound. RESULTS: Patients infected with S. japonicum had a much higher percentage of MDSCs among peripheral blood mononuclear cells (PBMCs) than the healthy control. Regarding subpopulations of MDSCs, the percentage of granulocytic myeloid-derived suppressor cells (G-MDSCs) was clearly increased. Correlation analysis showed that the absolute cell counts of T-cell subsets correlated negatively with the percentages of MDSCs and G-MDSCs among PBMCs. The percentage of G-MDSCs in PBMCs was also significantly higher in patients with liver fibrosis diagnosed by color doppler ultrasound (grade > 0), and the percentage of G-MDSCs in PBMCs and liver fibrosis grading based on ultrasound showed a positive correlation. CONCLUSION: S. japonicum infection contributes to an increase in MDSCs, especially G-MDSCs, whose proliferation may inhibit the number of CD4+ T cells in peripheral blood. Meanwhile, there is a close relationship between proliferation of G-MDSCs and liver fibrosis in S. japonicum-infected patients.

Title: La prolifération des MDSC peut indiquer une réponse immunitaire des lymphocytes T CD4+ plus faible dans la schistosomiase japonica. Abstract: Contexte : Schistosoma japonicum est la principale espèce de Schistosoma répandue en Chine. Les cellules myéloïdes suppressives (MDSC) sont des cellules immunorégulatrices importantes et se développent généralement lors d'une infection parasitaire, mais il existe peu de recherches sur les MDSC dans l'infection à Schistosoma. Méthodes : Cinquante-six patients infectés par S. japonicum ont été inclus dans cette étude. Les MDSC, les pourcentages et les nombres absolus des sous-ensembles de lymphocytes, notamment les lymphocytes T CD3+, les lymphocytes T CD4+, les lymphocytes T CD8+, les lymphocytes B et les cellules tueuses naturelles (NK) ont été détectés par cytométrie en flux. Le degré de fibrose hépatique a été déterminé par échographie Doppler couleur. Résultats : Les patients infectés par S. japonicum présentaient un pourcentage beaucoup plus élevé de MDSC parmi les cellules mononucléées du sang périphérique (CMSP) que les patients sains. En ce qui concerne les sous-populations de MDSC, le pourcentage de cellules suppressives granulocytaires dérivées de myéloïdes (G-MDSC) était augmenté de manière évidente. L'analyse de corrélation a montré que le nombre absolu des cellules des sous-ensembles de lymphocytes T était en corrélation négative avec les pourcentages de MDSC et de G-MDSC parmi les CMSP. Le pourcentage de G-MDSC dans les CMSP était également significativement plus élevé chez les patients présentant une fibrose hépatique diagnostiquée par échographie Doppler couleur (grade > 0), et le pourcentage de G-MDSC dans les CMSP et le classement de la fibrose hépatique basé sur l'échographie ont montré une corrélation positive. Conclusion : L'infection à S. japonicum contribue à une augmentation des MDSC, notamment des G-MDSC, dont la prolifération pourrait inhiber le nombre de lymphocytes T CD4+ dans le sang périphérique. Parallèlement, il existe une relation étroite entre la prolifération des G-MDSC et la fibrose hépatique chez les patients infectés par S. japonicum.

Immunization with the glutathione S-transferase Sj26GST with Chi-CpG NP against Schistosoma japonicum in mice.

Schistosomiasis caused by Schistosoma japonicum (S. japonicum) is a major public health problem in the Philippines, China and Indonesia. In this study, the immunopotentiator CpG-ODN was encapsulated within chitosan nanoparticles (Chi NPs) to create a combination adjuvant (Chi-CpG NP). This approach was employed to enhance the immunogenicity of 26 kDa glutathione S-transferase (Sj26GST) from S. japonicum through intranasal immunization. The results demonstrated higher levels of specific anti-Sj26GST antibodies and Sj26GST-specific splenocyte proliferation compared to mice that were immunized with Sj26GST + Chi-CpG NP. Cytokine analysis of splenocytes revealed that the Sj26GST + Chi-CpG NP induced a slight Th1-biased immune response, with increased production of IFN-γ by CD4+ T-cells in the spleen. Subsequently, mice were intradermally inoculated with 1 × 107 organisms in the Coeliac cavity. The bacterial organ burden detected in the liver of immunized mice suggested that Sj26GST + Chi-CpG NP enhances protective immunity to inhibit S. japonicum colonization. Therefore, Sj26GST + Chi-CpG NP vaccination enhances Sj26GST-specific immunogenicity and provides protection against S. japonicum.

An insight into the gut microbiota after Schistosoma japonicum eggs immunization in an experimental ulcerative colitis model.

Schistosome infection and schistosome-derived products have been implicated in the prevention and alleviation of inflammatory bowel disease by manipulating the host immune response, whereas the role of gut microbiota in this protective effect remains poorly understood. In this study, we found that the intraperitoneal immunization with Schistosoma japonicum eggs prior to dextran sulfate sodium (DSS) application significantly ameliorated the symptoms of DSS-induced acute colitis, which was characterized by higher body weight, lower disease activity index score and macroscopic inflammatory scores. We demonstrated that the immunomodulatory effects of S. japonicum eggs were accompanied by an influence on gut microbiota composition, abundance, and diversity, which increased the abundance of genus Turicibacter, family Erysipelotrichaceae, phylum Firmicutes, and decreased the abundance of genus Odoribacter, family Marinifilaceae, order Bacteroidales, class Bacteroidia, phylum Bacteroidota. In addition, Lactobacillus was identified as a biomarker that distinguishes healthy control mice from DSS-induced colitis mice. The present study revealed the importance of the gut microbiota in S. japonicum eggs exerting protective effects in an experimental ulcerative colitis (UC) model, providing an alternative strategy for the discovery of UC prevention and treatment drugs.

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.

The Protective Effect of the Soluble Egg Antigen of Schistosoma japonicum in A Mouse Skin Transplantation Model.

Background: Organ transplantation is currently an effective method for treating organ failure. Long-term use of immunosuppressive drugs has huge side effects, which severely restricts the long-term survival of patients. Schistosoma can affect the host's immune system by synthesizing, secreting, or excreting a variety of immunomodulatory molecules, but its role in transplantation was not well defined. In order to explore whether Schistosoma-related products can suppress rejection and induce long-term survival of the transplant, we used soluble egg antigen (SEA) of Schistosoma japonicum in mouse skin transplantation models. Materials and methods: Each mouse was intraperitoneally injected with 100 µg of SEA three times a week for four consecutive weeks before allogenic skin transplant. Skin transplants were performed on day 0 to observe graft survival. Pathological examination of skin grafts was conducted 7 days post transplantation. The skin grafts were subjected to mRNA sequencing. Bioinformatics analysis was conducted and the expression of hub genes was verified by qPCR. Flow cytometry analysis was performed to evaluate the immune status and validate the results from bioinformatic analysis. Results: The mean survival time (MST) of mouse skin grafts in the SEA-treated group was 11.67 ± 0.69 days, while that of the control group was 8.00 ± 0.36 days. Pathological analysis showed that Sj SEA treatment led to reduced inflammatory infiltration within skin grafts 7 days after allogenic skin transplantation. Bioinformatics analysis identified 86 DEGs between the Sj SEA treatment group and the control group, including 39 upregulated genes and 47 downregulated genes. Further analysis revealed that Sj SEA mediated regulation on cellular response to interferon-γ, activation of IL-17 signaling and chemokine signaling pathways, as well as cytokine-cytokine receptor interaction. Flow cytometry analysis showed that SEA treatment led to higher percentages of CD4+IL-4+ T cells and CD4+Foxp3+ T cells and decreased CD4+IFN-γ+ T cells in skin transplantation. Conclusion: Sj SEA treatment suppressed rejection and prolonged skin graft survival by regulating immune responses. Sj SEA treatment might be a potential new therapeutic strategy to facilitate anti-rejection therapy and even to induce tolerance.

miR-181a regulates the host immune response against Schistosoma japonicum infection through the TLR4 receptor pathway.

BACKGROUND: Schistosomiasis japonica is a serious zoonotic parasitic disease. Preliminary studies have shown that the expression of microRNA-181a (miR-181a) in the liver, lung and spleen tissues of susceptible host BALB/c mice and resistant host reed vole (Microtus fortis) 10 days post-infection (dpi) with Schistosoma japonicum was significantly different from pre-infection levels. This difference suggests the possibility that miR-181a expression may be related to the regulation of the hosts' early immune response against S. japonicum infection and thereby affect the development and survival of parasites in their final hosts. METHODS: BALB/c mice, M. fortis, Toll-like receptor 4 (TLR4)-deficient mice and wild-type mice (C57BL/6) were infected with S. japonicum, and differences in miR-181a expression between BALB/c mice and M. fortis over different time points post-infection (0, 3, 7, 10 and 14 dpi) were compared. MiR-181a mimic, miR-181a inhibitor and irrelevant miRNA, as well as lipopolysaccharide (LPS), a TLR4 receptor ligand, were used to transfect mouse RAW264.7 macrophages. The expression levels of the TLR4 pathway-related cytokines interleukin (IL)-1ß, tumor necrosis factor α (TNF-α) and IL-6 were detected by quantitative PCR analysis. RESULTS: The expression of miR-181a was significantly upregulated in the serum and liver of mice infected with S. japonicum and downregulated in the serum and liver of M. fortis. T-helper cell (Th1)-type cytokines, such as TNF-α, IL-6 and IL-1ß, and Th2-type cytokines, such as IL-10 and IL-4, were differentially expressed in M. fortis and BALB/c mice in the early stage of infection. The expression level of miR-181a in the serum was threefold higher in TLR4-deficient mice than in wild-type mice 10 dpi with S. japonicum. The expression of IL-1ß, TNF-α and IL-6 decreased in RAW264.7 cells transfected with miR-181a mimic and increased in cells transfected with miR-181a inhibitor. miR-181a expression was downregulated and the expressions of TLR4 and three TLR4 pathway-related cytokines (IL-1ß, IL-6, and TNF-α) were upregulated in RAW264.7 macrophages stimulated with the TLR4 receptor ligand LPS. CONCLUSION: These results suggest the possibility of mutual regulation between miR-181a and the TLR4 signaling pathway during S. japonicum infection. miR-181a may regulate the expression of pro-inflammatory factors through the TLR4 receptor pathway and participate in the immunomodulatory effect of anti-S. japonicum infection.

Ikzf2 Regulates the Development of ICOS+ Th Cells to Mediate Immune Response in the Spleen of S. japonicum-Infected C57BL/6 Mice.

Background: Th cells (helper T cells) have multiple functions in Schistosoma japonicum (S. japonicum) infection. Inducible co-stimulator (ICOS) is induced and expressed in activated T lymphocytes, which enhances the development of B cells and antibody production through the ICOS/ICOSL pathway. It remains unclear about the role and possible regulating mechanism of ICOS+ Th cells in the spleen of S. japonicum-infected C57BL/6 mice. Methods: C57BL/6 mice were infected with cercariae of S. japonicum through the abdomen. The expression of ICOS, activation markers, and the cytokine production on CD4+ ICOS+ Th cells were detected by flow cytometry (FCM) and quantitative real-time PCR (qRT-PCR). Moreover, the differentially expressed gene data of ICOS+ and ICOS- Th cells from the spleen of infected mice were obtained by mRNA sequencing. Besides, Western blot and chromatin immunoprecipitation (ChIP) were used to explore the role of Ikzf2 on ICOS expression. Results: After S. japonicum infection, the expression of ICOS molecules gradually increased in splenic lymphocytes, especially in Th cells (P < 0.01). Compared with ICOS- Th cells, more ICOS+ Th cells expressed CD69, CD25, CXCR5, and CD40L (P < 0.05), while less of them expressed CD62L (P < 0.05). Also, ICOS+ Th cells expressed more cytokines, such as IFN-γ, IL-4, IL-10, IL-2, and IL-21 (P < 0.05). RNA sequencing results showed that many transcription factors were increased significantly in ICOS+ Th cells, especially Ikzf2 (P < 0.05). And then, the expression of Ikzf2 was verified to be significantly increased and mainly located in the nuclear of ICOS+ Th cells. Finally, ChIP experiments and dual-luciferase reporter assay confirmed that Ikzf2 could directly bind to the ICOS promoter in Th cells. Conclusion: In this study, ICOS+ Th cells were found to play an important role in S. japonicum infection to induce immune response in the spleen of C57BL/6 mice. Additionally, Ikzf2 was found to be one important transcription factor that could regulate the expression of ICOS in the spleen of S. japonicum-infected C57BL/6 mice.

Schistosoma japonicum peptide SJMHE1 inhibits acute and chronic colitis induced by dextran sulfate sodium in mice.

BACKGROUND: Harnessing helminth-based immunoregulation is a novel therapeutic strategy for many immune dysfunction disorders, including inflammatory bowel diseases (IBDs). We previously identified a small molecule peptide from Schistosoma japonicum and named it SJMHE1. SJMHE1 can suppress delayed-type hypersensitivity, collagen-induced arthritis and asthma in mice. In this study, we assessed the effects of SJMHE1 on dextran sulfate sodium (DSS)-induced acute and chronic colitis. METHODS: Acute and chronic colitis were induced in C57BL/6 mice by DSS, following which the mice were injected with an emulsifier SJMHE1 or phosphate-buffered saline. The mice were then examined for body weight loss, disease activity index, colon length, histopathological changes, cytokine expression and helper T (Th) cell subset distribution. RESULTS: SJMHE1 treatment significantly suppressed DSS-induced acute and chronic colitis, improved disease activity and pathological damage to the colon and modulated the expression of pro-inflammatory and anti-inflammatory cytokines in splenocytes and the colon. In addition, SJMHE1 treatment reduced the percentage of Th1 and Th17 cells and increased the percentage of Th2 and regulatory T (Treg) cells in the splenocytes and mesenteric lymph nodes of mice with acute colitis. Similarly, SJMHE1 treatment upregulated the expression of interleukin-10 (IL-10) mRNA, downregulated the expression of IL-17 mRNA and modulated the Th cell balance in mice with chronic colitis. CONCLUSIONS: Our data show that SJMHE1 provided protection against acute and chronic colitis by restoring the immune balance. As a small molecule, SJMHE1 might be a novel agent for the treatment of IBDs without immunogenicity concerns.

Performance of the point-of-care circulating cathodic antigen test in the diagnosis of schistosomiasis japonica in a human cohort from Northern Samar, the Philippines.

BACKGROUND: Zoonotic schistosomiasis, caused by Schistosoma japonicum, remains a major public health problem in the Philippines. This study aimed to evaluate the commercially available rapid diagnostic point-of-care circulating cathodic antigen (POC-CCA) test in detecting individuals infected with S. japonicum in a human cohort from an endemic area for schistosomiasis japonica in the Philippines. METHODS: Clinical samples were collectedin 18 barangays endemic for S. japonicum infection in Laoang and Palapag municipalities, Northern Samar, the Philippines, in 2015. The presence of CCA in filter-concentrated urine samples (n = 412) was evaluated using the commercial kits and the results were converted to images, which were further analyzed by ImageJ software to calculate R values. The diagnostic performance of the immunochromatographic POC-CCA test was compared using the Kato-Katz (KK) procedure, in-house enzyme-linked immunosorbent assays (ELISAs) and droplet digital (dd) PCR assays as reference. RESULTS: The POC-CCA test was able to detect S. japonicum-infected individuals in the cohort with an eggs per gram of faeces (EPG) more than or equal to 10 with sensitivity/specificity values of 63.3%/93.3%. However, the assay showed an inability to diagnose schistosomiasis japonica infections in all cohort KK-positive individuals, of which the majority had an extremely low egg burden (EPG: 1-9). The prevalence of S. japonicum infection in the total cohort determined by the POC-CCA test was 12.4%, only half of that determined by the KK method (26.2%). When compared with the ELISAs and ddPCR assays as a reference, the POC-CCA assay was further shown to be a test with low sensitivity. Nevertheless, the assay exhibited significant positive correlations with egg burden determined by the KK technique and the target gene copy number index values determined by the ddPCR assays within the entire cohort. CONCLUSIONS: By using in silico image analysis, the POC-CCA cassette test could be converted to a quantitative assay to avoid reader-variability. Because of its low sensitivity, the commercially available POC-CCA assay had limited potential for determining the status of a S. japonicum infection in the target cohort. The assay should be applied with caution in populations where schistosome parasites (especially S. japonicum) are present at low infection intensity.

MiR-130a-3p Alleviates Liver Fibrosis by Suppressing HSCs Activation and Skewing Macrophage to Ly6Clo Phenotype.

Emerging evidences have highlighted the crucial role of microRNAs (miRNAs) in the liver cirrhosis, but the relationship between miR-130a-3p and liver cirrhosis is not entirely clear. As we all know, schistosomiasis, as one of the zoonoses, can lead to liver cirrhosis when it advances. In this study, we investigated the biological functions of miR-130a-3p on the liver fibrosis of schistosomiasis in vivo and in vitro. The mice infected with Schistosoma japonicum (S. japonicum) were treated with lentivirus vector (LV)-miR-130a-3p by hydrodynamic injection through the tail vein. Our findings showed significantly decreased expression of miR-130a-3p both in the serum of patients with cirrhosis and in the liver of mice infected with S. japonicum. The results showed that LV-miR-130a-3p could effectively enter into the liver and alleviate liver granulomatous inflammation and collagen deposition. Simultaneously, LV-miR-130a-3p-promoted macrophages presented the Ly6Clo phenotype, concomitant with the decreased expression of the tissue inhibitor of metalloproteinases (TIMP) 1, and increased the expression of matrix metalloproteinase (MMP) 2, which contributed to the dissolution of collagen. Furthermore, overexpression of miR-130a-3p not only inhibited the activation and proliferation of hepatic stellate cells (HSCs) but also induced the apoptosis of HSCs. In addition, we also confirmed that miR-130a-3p enables to bind with mitogen-activated protein kinase (MAPK) 1 and transforming growth factor-beta receptors (TGFBR) 1 and TGFBR2 genes and inhibit the expressions of these genes. Our findings suggested that miR-130a-3p might represent as the potential candidate biomarker and therapeutic target for the prognosis identification and treatment of schistosomiasis liver fibrosis.

Comparison of point-of-care test and enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies in the diagnosis of human schistosomiasis japonica.

OBJECTIVE: Schistosomiasis japonica is an important helminthic disease in Asia. Sensitive and accurate diagnostic tools are indispensable for clinical diagnosis, screening infection and monitoring its control. In this study, we developed an immunochromatographic test (Sj-ICT) to detect anti-Schistosoma japonicum immunoglobulin G antibodies in human sera. METHODS: Somatic extract from adult S. japonicum was used as an antigen. The Sj-ICT was developed and optimized as a point-of-care test. All 214 human serum samples were evaluated for diagnostic usefulness and comparison with an enzyme-linked immunosorbent assay (ELISA). RESULTS: The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy of the Sj-ICT were 90.8%, 87.9%, 86.4%, 91.9% and 89.3%, respectively. For ELISA the values were respectively 91.8%, 87.9%, 86.5%, 92.7% and 89.7%. The concordance between both methods was 86.4 % (Cohen's kappa value = 0.729). CONCLUSIONS: The immunochromatographic test kit developed can support clinical diagnosis and large-scale surveys in endemic areas without requiring additional facilities or ancillary supplies.

Revisiting the Schistosoma japonicum life cycle transcriptome for new insights into lung schistosomula development.

Schistosome parasites are complex trematode blood flukes responsible for the disease schistosomiasis; a global health concern prevalent in many tropical and sub-tropical countries. While established transcriptomic databases are accessed ad hoc to facilitate studies characterising specific genes or gene families, a more comprehensive systematic updating of gene annotation and survey of the literature to aid in annotation and context is rarely addressed. We have reanalysed an online transcriptomic dataset originally published in 2009, where seven life cycle stages of Schistosoma japonicum were examined. Using the online pathway analysis tool Reactome, we have revisited key data from the original study. A key focus of this study was to improve the interpretation of the gene expression profile of the developmental lung-stage schistosomula, since it is one of the principle targets for worm elimination. Highly enriched transcripts, associated with lung schistosomula, were related to a number of important biological pathways including host immune evasion, energy metabolism and parasitic development. Revisiting large transcriptomic databases should be considered in the context of substantial new literature. This approach could aid in the improved understanding of the molecular basis of parasite biology. This may lead to the identification of new targets for diagnosis and therapies for schistosomes, and other helminths.

A novel fluorescence immunochromatographic assay strip for the diagnosis of schistosomiasis japonica.

BACKGROUND: Schistosomiasis japonica is a severe zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. Surveillance and diagnosis play key roles in schistosomiasis control; however, current techniques for the surveillance and diagnosis of the disease have limitations. In this study, we developed a novel fluorescence immunochromatographic assay (FICA) strip to detect anti-Schistosoma japonicum antibodies in host serum. METHODS: A FICA strip was developed for the diagnosis of Schistosoma japonicum in domestic animals. Streptococcus protein G (SPG) and soluble egg antigen (SEA) were transferred onto a nitrocellulose (NC) membrane to form the control line (C) and the test line (T), respectively. With fluorescence activity as well as binding activity to multispecies IgG, the recombinant protein rSPG-RFP was expressed and employed as an antibody indicator in the FICA strips. RESULTS: The dual gene fusion plasmid was verified by PCR and restriction enzyme digestion. The expressed recombinant protein was 39.72 kDa in size, which was consistent with the predicted molecular weight. The western blot results showed binding activity between rSPG-RFP and IgGs from different hosts. Fluorescence microscopy also showed the fluorescence activity of the protein present. The affinity constant (Ka) values of rSPG-RFP with rabbit, donkey, mouse and goat IgG were 1.9 × 105, 4.1 × 105, 1.7 × 105 and 5.4 × 105, respectively. Moreover, based on the recombinant protein, the test strip for detecting S. japonicum in buffaloes could distinguish positive from negative serum. The lower limit of detection of the FICA strip was 1:10,000. Compared with ELISA, the FICA strips exhibited similar results in the diagnosis of infection in clinical bovine serum samples, with a kappa value of 0.9660 and P < 0.01. The cross-reactivities of the FICA strips with Haemonchus contortus and Schistosoma turkestanicum (30.15% and 91.66%, respectively) were higher than those of ELISA (26.98% and 87.5%, respectively). CONCLUSIONS: Based on the rSPG-RFP protein that we developed, strip detection can be completed within 15 min. Heightened sensitivity allows the strip to accurately identify schistosome antibodies in serum. In conclusion, this method is convenient, feasible, rapid and effective for detecting S. japonicum.

Reviews and advances in diagnostic research on Schistosoma japonicum.

Schistosomiasis is an acute and chronic parasitic disease caused by blood flukes (trematode worms) of the genus Schistosoma. Schistosoma japonicum (S. japonicum) infection has decreased significantly in prevalence and intensity of infection in China. However, this disease still remains a serious public health problem in some endemic areas of the Philippines and Indonesia. Thus, more accurate and sensitive methods are much needed for further control of this disease. Here, we review the research progress in techniques for the diagnosis of S. japonicum infection.

Mechanism by which the combination of SjCL3 and SjGAPDH protects against Schistosoma japonicum infection.

A vaccine is an important method to control schistosomiasis. Molecules related to lung-stage schistosomulum are considered potential vaccine candidates. We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cathepsin L3 (CL3) displayed differential expression in the lung-stage schistosomula of Schistosoma japonicum cocultured with host cells. In the present study, we prepared the two proteins and detected the protective effects of SjGAPDH by immunizing mice with this protein alone and in combination with SjCL3 with or without Freund's adjuvant. Then, we investigated the possible mechanisms underlying S. japonicum infection. The results showed that vaccination of adjuvanted SjGAPDH decreased the worm burden (37.8%) and egg load (38.1%), and the combination of adjuvanted SjGAPDH and SjCL3 further decreased the worm burden (65.6%) and egg load (70.9%) during Schistosoma japonicum infection. However, the immunization of a combination of adjuvant-free SjGAPDH and SjCL3 displayed a lower protective effect (< 15%) than those of the adjuvanted SjCL3, the adjuvanted SjGAPDH, and a combination of adjuvanted SjGAPDH and SjCL3. Flow cytometric results showed that the frequency of regulatory T cells (Tregs) was lower (P < 0.05) in the group with adjuvanted SjGAPDH and SjCL3 (2.61%) than the remaining groups. The enzyme-linked immunosorbent assay (ELISA) results indicated that except for the uninfected and infected control groups, the remaining groups displayed a Th1-type shift in immune responses. These results showed the immunization of SjGAPDH resulted in partial protection (approximately 38%); inoculation with a combination of SjCL3 and SjGAPDH in Freund's adjuvant resulted in a high immunoprotective effect (> 65%) against Schistosoma japonicum infection in mice, which was possibly caused by the reduced percentage of Tregs and a Th1-type shift in immune responses; and SjCL3 has no adjuvant-like effect, dissimilar to SmCL3.

Hepatocyte CD1d protects against liver immunopathology in mice with schistosomiasis japonica.

Schistosomiasis is a neglected tropical disease with over 250 million people infected worldwide. The main clinically important species Schistosoma mansoni (S. mansoni) and Schistosoma japonicum (S. japonicum) cause inflammatory responses against tissue-trapped eggs, resulting in formation of granulomas mainly in host liver. Persistent granulomatous response results in severe fibrosis in the liver, leading to irreversible impairment of the liver and even death of the host. CD1d, a highly conserved MHC class I-like molecule, is expressed by both haematopoietic and non-haematopoietic cells. CD1d on antigen-presenting cells (APCs) of haematopoietic origin presents pathogen-derived lipid antigens to natural killer T (NKT) cells, which enables them to rapidly produce large amounts of various cytokines and facilitate CD4+ T helper (Th) cell differentiation upon invading pathogens. Noteworthy, hepatocytes of non-haematopoietic origin have recently been shown to be involved in maintaining liver NKT cell homeostasis through a CD1d-dependent manner. However, whether hepatocyte CD1d-dependent regulation of NKT cell homeostasis also modulates CD4+ Th cell responses and liver immunopathology in murine schistosomiasis remains to be addressed. Here, we show in mice that CD1d expression on hepatocytes was decreased dramatically upon S. japonicum infection, accompanied by increased NKT cells, as well as upregulated Th1 and Th2 responses. Overexpression of CD1d in hepatocytes significantly decreased local NKT numbers and cytokines (IFN-γ, IL-4, IL-13), concomitantly with downregulation of both Th1 and Th2 responses and alleviation in pathological damage in livers of S. japonicum-infected mice. These findings highlight the potential of hepatocyte CD1d-targeted therapies for liver immunopathology control in schistosomiasis.

A longitudinal study reveals the alterations of the Microtus fortis colonic microbiota during the natural resistance to Schistosoma japonicum infection.

The gut microbiota has been demonstrated to associate with protection against helminth infection and mediate via microbial effects on the host humoral immunity. As a non-permissive host of Schistosoma japonicum, the Microtus fortis provides an ideal animal model to be investigated, because of its natural self-healing capability. Although researches on the systemic immunological responses have revealed that the host immune system contributes a lot to the resistance, the role of gut microbiome remains unclear. In this study, we exposed the M. fortis to the S.japonicum infection, carried out a longitudinal research (uninfected control, infected for 7 days, 14 days, 21 days, and 31 days) on their colonic microbiota based on the 16S rRNA gene amplicon sequencing. The bacterial composition disclosed a disturbance-recovery alteration followed by the resistance to S. japonicum. The alpha diversity of colon microbiota was reduced after the infection, but it gradually recovered along with self-healing process. Further LEfSe analysis revealed that phyla shifted from Firmicutes to Bacteroidetes, which were mainly driven by an increase of Ruminococcaceae and a depletion of Muribaculaceae in the family level along the Control-Infection-Recovery (CIR) process. We identified a temporary blooming of Lactobacillaceae and Lactobacillus in the mid infection stage (D14). As a recognized probiotics repository, we speculate the increased abundance of Lactobacillaceae in M. fortis colonic microbiota might relate to the natural resistance to the schistosome. Besides, potential microbial functions were also significantly changed in the resistance process. These results demonstrate the remarkable alterations of reed vole colonic microbiota in both community structure and potential functions along with the resistance to S. japonicum infection. The identified microbial biomarkers might offer new ways for drug development to conquer human schistosomiasis.

Therapeutic potential of fucoidan in the reduction of hepatic pathology in murine schistosomiasis japonica.

BACKGROUND: Hepatic granuloma formation and fibrosis as the consequence of tissue entrapped eggs produced by female schistosomes characterize the pathology of Schistosoma japonicum infection. It has been proposed that fucoidan, a sulfated polysaccharide existing naturally in brown seaweed Fucus vesiculosus, plays a diversified role to perform immunomodulatory activities. However, whether fucoidan functions in the host hepatic pathology is unknown and identifying the potential mechanism that is responsible for hepatic improvement is still necessary. METHODS: We evaluated the hepatic pathology from S. japonicum-infected mice after treatment with fucoidan. qRT-PCR and immunofluorescence were used to detect the pro- or anti-inflammatory factors and the phosphorylated p65 in the livers. In addition, flow cytometry was also performed to investigate the T cell subsets in the S. japonicum-infected mice after treatment with fucoidan, and functional molecules relatively specific to Treg cells were detected in vitro. Furthermore, macrophages were treated with fucoidan in vitro and to detect the inflammatory cytokines. RESULTS: Treatment with fucoidan significantly reduced the hepatic granuloma size and fibrosis response during S. japonicum infection. The attenuated phospho-p65 protein levels and the mRNA levels of pro-inflammatory cytokines (IL-6, IL-12 and TNF-α) were observed in the livers from fucoidan-treated S. japonicum-infected mice; however, the mRNA levels of anti-inflammatory cytokines (IL-4 and IL-13) were increased. In addition, the infiltration of Treg cells was significantly enhanced both in the livers and spleens from fucoidan-treated S. japonicum-infected mice. Consistent with this, the mRNA levels of IL-10 and TGF-ß were dramatically increased in the livers from S. japonicum-infected mice after fucoidan treatment. Furthermore, in vitro stimulated splenocytes with fucoidan resulted in increasing Treg cells in splenocytes as well as the functional expression of CC chemokine receptor type 4 (CCR4) and CXC chemokine receptor type 5 (CXCR5) in Treg cells. Additionally, fucoidan promoted the mRNA levels of IL-4 and IL-13 in macrophages. CONCLUSIONS: These findings suggest an important role of natural fucoidan in reducing hepatic pathology in the progress of S. japonicum infection with a stronger Treg response, which may reveal a new potential therapeutic strategy for hepatic disease caused by parasitic chronic infection.

Analyses of the expression, immunohistochemical properties and serodiagnostic potential of Schistosoma japonicum peroxiredoxin-4.

BACKGROUND: Schistosoma japonicum, which inhabits the mesenteric vein of the mammalian hosts for about 20 to 30 years, is subjected to the oxidative stresses from the host defense mechanism during their intra-mammalian stages. To counteract this host immune attack, the parasite utilizes their antioxidant system for survival inside the host. Peroxiredoxins (Prxs), thiol-specific antioxidant proteins, play an essential role for protecting the parasite against oxidative stress by reducing hydrogen peroxide to water. Only three types of 2-Cys Prxs have been previously characterized in S. japonicum whereas a fourth Prx has been identified for Schistosoma mansoni as Prx-4. A sequence coding homologous to this gene in the S. japonicum database was identified, characterized and expressed as recombinant SjPrx-4 protein (rSjPrx-4). Furthermore, rSjPrx-4 was evaluated in this study for its diagnostic potentials in detecting S. japonicum infection in humans. RESULTS: The gene found in the parasite genome contained 2 active-site cysteines with conserved sequences in the predicted amino acid (AA) sequence and showed 75% identity with that of the previously characterized Prx (TPx-1) of S. japonicum. The gene was expressed in different stages of schistosome life-cycle with highest transcription level in the adult male. The gene was cloned into a plasmid vector and then transfected into Escherichia coli for expression of rSjPrx-4. Anti-rSjPrx-4 mouse sera recognized native SjPrx-4 in egg and adult worm lysate by western blotting. The result of a mixed function oxidation assay in which rSjPrx-4 prevented the nicking of DNA from hydroxyl radicals confirmed its antioxidant activity. Subsequently, immunolocalization analysis showed the localization of SjPrx-4 inside the egg, on the tegument and in the parenchyma of the adult worm. Enzyme-linked immunosorbent assay results showed that rSjPrx-4 has 83.3% sensitivity and 87.8% specificity. Its diagnostic potential was further evaluated in combination with recombinant SjTPx-1 protein, yielding an improved sensitivity and specificity of 90% and 92.7%, respectively. CONCLUSIONS: These results suggest that SjPrx-4 plays a role as an antioxidant dealing with oxidative stresses of S. japonicum, and its diagnostic potential improved by coupling it with SjTPx-1 is a proof for developing a serological test with better diagnostic performance for human schistosomiasis.