High prevalence of Schistosoma japonicum by perfusion in naturally exposed water buffalo in a region of the Philippines endemic for human schistosomiasis.

In the past decade, ecological surveys emphasized rats and dogs as the most significant animal reservoirs for Schistosoma japonicum (S.j) in the Philippines. However, recent studies demonstrated 51-91% prevalence of schistosomiasis among water buffalo using qPCR in the Sj endemic regions in the Philippines. In order to resolve the inconsistency of reported surveys regarding Sj endemicity among carabao, a domestic water buffalo that is the most important draught animal, we introduced 42 schistosome negative water buffalo to Macanip, Jaro municipality, Leyte, the Philippines, a subsistence rice-farming village that has been the focus of schistosomiasis japonica studies of our group for the past 20 years. We conducted perfusion to the remaining 34 buffalo that survived 10 months of nature exposure and Typhoon Haiyan. Thirty-three water buffalo were found to be positive with at least 1 pair of worms from the mesenteric vein. The infection rate is 97%, with the worm burden of 94 (95% confidence interval, 49-138 worms) worms. To our knowledge, this is the first report about S. japonicum worm burden in naturally infected water buffalo in the Philippines. The fact that with less than one-year of exposure, in this human schistosomiasis endemic area, only 1 out of 34 water buffalo was uninfected is striking. Urgent attention is needed for a cost-effective technique for monitoring Sj infection in animals and humans. Meanwhile, intervention implementation, including water buffalo treatment and vaccination, should be taken into consideration.

Vaccination with recombinant paramyosin in Montanide ISA206 protects against Schistosoma japonicum infection in water buffalo.

BACKGROUND: Schistosomiasis japonica is a zoonosis and presents significant public health problems in China and the Philippines. Vaccines targeting domestic animals constitute attractive control measures. METHODS: We conducted three vaccine trials to evaluate the protective efficacy of recombinant full-length paramyosin (rSj97) in water buffalo. Animals were immunized with 3 doses of rSj97 adjuvanted with ISA206 at 250µg/dose or 500µg/dose at 4wk intervals before challenge with 1000 Schistosoma japonicum cercariae. The primary outcome was worm burden assessed by portal perfusion 8-10weeks post challenge. Safety measures included weight, temperature, body condition score, hemogram and routine assays for hepatic and renal function. RESULTS: The three-dose regimen was well tolerated in all three trials. In the first trial, vaccinated buffalo had 51.5% lower worm burden post challenge compared to controls. In the second trial, buffalo immunized with 500µg/dose of rSj97 had 57.8% lower worm burden compared to controls (p=0.026). A similar but not significant reduction (60.9%) was observed with animals administered with 250ug rSj97/dose. In the third trial, buffalo immunized with a 500µg/dose of rSj97 had 57.8% lower worm burden compared to controls (p=0.014). CONCLUSIONS: These findings indicated that rSj97 is a safe and promising vaccine candidate for schistosomiasis japonica in water buffalo.

Nested-PCR assay for detection of Schistosoma japonicum infection in domestic animals.

BACKGROUND: Schistosomiasis japonica is a common zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. The prevalence and infectivity of this disease in domestic animals in China have significantly decreased and, for this reason, diagnostics with a higher sensitivity have become increasingly necessary. It was reported that polymerase chain reaction (PCR)-based methods could be used to detect schistosome infection in humans and animals and presented a high sensitivity and specificity. The present study aimed to develop a PCR-based method for detection of Schistosoma japonicum infection in domestic animals. METHODS: A specific nested-PCR assay was developed to detect S. japonicum infection in domestic animals via amplification of a 231-bp DNA fragment of retrotransposon SjR2. The developed assay was first used in sera and dry blood filter paper (DBFP) from goats and buffaloes at different time points of infection. Then, 78 DBFPs from 39 artificially-infected bovines at 14 and 28 days post-infection and 42 DBFPs from schistosome-negative bovines from the city of Huangshan in the Anhui province were used to evaluate the diagnostic validity. Furthermore, this assay was used to detect S. japonicum infection in domestic animals in Dongzhi and Wangjiang counties. RESULTS: The expected PCR product was detected in eggs and adult worms of S. japonicum and blood samples from S. japonicum-infected goats and water buffaloes, but not from Fasciola and Haemonchus contortus worms. The nested-PCR assay could detect the target S. japonicum DNA in DBFPs from goats and buffaloes after day 3 post-infection. The sensitivity in buffaloes at 14 and 28 days post-infection was 92.30% (36/39) and 100% (39/39), respectively. The specificity was 97.60% (41/42). The positivity rates in Dongzhi and Wangjiang counties were 6.00% and 8.00% in bovines and 22.00% and 16.67% in goats, respectively. The positivity rates in goats in both counties were higher than those in bovines with a significant difference in Dongzhi County but not in Wangjiang County (P < 0.05 and P = 0.23, respectively). CONCLUSIONS: Our results suggest that the developed nested-PCR assay may be used for the diagnosis of S. japonicum infection in domestic animals, and the control of S. japonicum infection in goats should be paid more attention.

[Preliminary study on pro-apoptotic gene BAD of Schistosoma japonicum].

OBJECTIVE: To understand the characteristics of pro-apoptotic gene SjBAD of Schistosoma japonicum, such as its biology, immunology, and transcriptional expression, and evaluate its potential of the recombinant protein as a vaccine candidate for schistosomiasis. METHODS: SjBAD was amplified by PCR and subeloned into a pET-28a(+) vector, and the recombinant plasmid was transformed into competent E. coli BL21 for producing recombinant protein. The expressions of SjBAD in different development stages of schistosomula and 42-day male and female worms were determined by real-time PCR. The immunogenicity of the recombinant protein was analyzed by Western blotting and ELISA. The potential of this protein as a vaccine candidate molecule was assessed by testing the worm reduction rate and liver egg reduction rate in the BALB/c mice immunized by the recombinant antigen SjBAD. RESULTS: SjBAD was successfully cloned, the recombinant plasmid pET-28a(+)-SjBAD was successfully expressed in E. coli, and the molecular weight of the recombinant protein was around 22 kDa. Western-blotting showed that the recombinant protein had good immunogenicity. The recombinant protein could induce high level of specific IgG antibodies in the BALB/c mice. SjBAD was expressed in all tested 7-, 14-, 21-, 28-, 35- and 42-day worms, and was highly expressed in 14-day schistosomula, while the expression level in 42-day male worms was higher than that in 42-day female worms. Two in- dependent animal trials showed that 30.82% and 27.87% worm reduction rates, as well as 42.52% and 45.84% liver eggs reduction rates were obtained in the rSjBAD vaccinated group compared with those of the blank control group (both P < 0.05). CONCLUSIONS: The proapoptotic gene SjBAD is successfully cloned and expressed. The gene is expressed in different development stages of S. japonicum. The rSjBAD vaccinated BALB/c mice can obtain a partial protective immunity against S. japonicum infection.

SjHSP70, a recombinant Schistosoma japonicum heat shock protein 70, is immunostimulatory and induces protective immunity against cercarial challenge in mice.

High levels of protective immunity can be induced in different animals immunized with radiation-attenuated (RA) Schistosoma cercariae or schistosomula. However, the schistosome-derived molecules responsible for the strong protective effect elicited by RA schistosome larvae have not been identified or characterized. The 70-kDa heat shock proteins of schistosomes are considered major immunogens, and may play an important role in stimulating high levels of innate and adaptive immune responses in an RA schistosome vaccine model. Here, we demonstrate the immunobiological functions of Schistosoma japonicum heat shock protein 70 (SjHSP70) by investigating its expression profile in RA-schistosomula-derived cells, evaluating the protection induced by recombinant SjHSP70 (rSjHSP70) against cercarial challenge, and assaying the humoral and cellular immune responses to rSjHSP70 in BALB/c and C57BL/6 mice. The expression of SjHSP70 on the surfaces of cells from RA or normal schistosomula was determined with flow cytometry. Its expression was significantly higher on early RA schistosomula cells than on the cells from normal parasites. The protection afforded both BALB/c and C57BL/6 mice vaccinated with rSjHSP70 alone, rSj22.6 (a membrane-anchoring protein of S. japonicum) alone, or a combination of rSj22.6 and rSjHSP70 without adjuvant was evaluated. rSjHSP70 alone induced the highest protective effect against S. japonicum cercarial challenge, followed by the rSj22.6 plus rSjHSP70 combination and then rSj22.6 alone, in both mouse strains. Like ISA206 adjuvant, rSjHSP70 enhanced the protective efficacy induced by rSj22.6 in the C57BL/6 mouse strain. Antigen-specific IgG1 and IgG2a responses were detected with enzyme-linked immunosorbent assays in mice immunized with rSjHSP70 alone, rSj22.6 alone, or the rSj22.6 plus rSjHSP70 combination. Immunization with rSjHSP70 or the rSj22.6 plus rSjHSP70 combination induced mixed Th1/Th2-type antibody responses in BALB/c mice and a Th2-type antibody response in C57BL/6 mice. The profiles of cytokine production by splenic lymphocytes in both strains of mice immunized with the antigens described above were detected in vitro using a Cytometric Bead Array. The profiles of the proinflammatory cytokines interferon γ, tumor necrosis factor α, interleukin 6 (IL-6), and IL-17A and the regulatory cytokine IL-10 induced by the rSj22.6 plus rSjHSP70 combination were similar to those induced by rSj22.6 emulsified with the ISA206 adjuvant control. Like the ISA206 adjuvant, rSjHSP70 protein enhanced the proinflammatory and Th2-type or regulatory cytokine production induced by the rSj22.6 antigen. These results indicate that SjHSP70 is exposed on the surfaces of cells from RA schistosomula, and that rSjHSP70 protein is a promising protective antigen with a potential adjuvant function. Thus, SjHSP70 protein might play a key role in the protective immunity elicited by the RA schistosome vaccine.

Elimination of schistosomiasis japonica from formerly endemic areas in mountainous regions of southern China using a praziquantel regimen.

Schistosomiasis japonica is a major public health problem in China. Domestic animals play a major role in the transmission of Schistosoma japonicum to humans. To better understand the epidemiology of schistosomiasis japonica in domestic animals in the mountainous areas of China, we performed a 5-year longitudinal study of schistosomiasis in cattle and horses in Yunnan Province from 2009 to 2013. We also performed a concurrent drug-based intervention study in three settlement groups in Yunnan Province aimed at developing an effective means of controlling transmission in this region. The prevalence of infection in cattle fluctuated between 1.67% and 3.05% from 2009 to 2011, and monthly treatments of schistosome-positive animals reduced the prevalence to 0% (P<0.05) from 2012 to 2013. Prior to the intervention, we found that schistosomiasis was prevalent from May to October, with the highest prevalence observed in June (10.00%). We surveyed for environmental schistosome contamination, and 94.29% of the miracidia found were from cattle. Our study showed that it is possible to eliminate schistosomiasis in domestic animals in the mountainous regions of China by monthly treating cattle and horses from schistosome-positive households from May to October.

[Development of colloidal gold immunochromatography assay strip for schistosomiasis diagnosis in domestic animals].

OBJECTIVE: To develop a quick and easy colloidal gold immunochromatography assay (GICA) strip for schistosomiasis diagnosis in domestic animals. METHODS: The reconstruction of Streptococcal Protein G (SPG) was designed and its gene was subcloned into plasmid pET-28a(+) to express in Escherichia coli. The recombinant SPG was purified and labeled with colloidal gold. The Schistosoma japonicum soluble egg antigen (SEA) and rSPG were blotted on the nitrocellulose membrane for the test line and control line respectively. The specificity, sensitivity and cross-reaction of the strip method were detected. RESULTS: The rSPG was successfully expressed and purified to label with colloidal gold. The colloidal gold immunochromatography assay strips were assembled and they could detect the sera of S. japonicum infected BALB/c mice, New Zealand white rabbits, buffalo and sheep successfully. Besides, the sensitivity of GICA strip was 100% in the sera of mice and the serum of rabbits with S. japonicum infection. The specificity was 100% in the serum of mice and the sera of rabbits with free of infection. The sensitivity was 100% in the sera of sheep with miracidia of S. japonicum hatching from the stool and the specificity was 88.46% in the sera of sheep without that. The sensitivity was 94.44% in the sera of buffalo with miracidia hatching from the stool and the specificity was 100% in the sera of buffalo without that. The cross-reaction rate was 5.88% in Paramphistomum. CONCLUSION: The GICA strip can successfully detect a variety of S. japonicum infected domestic animals and may be a useful tool for screening on a large scale in the endemic areas.

The molecular characterization and RNAi silencing of SjZFP1 in Schistosoma japonicum.

During development, Schistosoma japonicum undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Proteins containing zinc finger motifs usually play an important role in DNA recognition, RNA packaging, and transcriptional activation. In our current study, we cloned the open reading frame (ORF) of SjZFP1 of S. japonicum, which encodes a zinc finger protein. We analyzed the complementary DNA (cDNA) sequence of SjZFP1 and examined the expression of SjZFP1 messenger RNA (mRNA) at various developmental stages. We also tested the effects of RNA interference (RNAi) silencing on worm burden, spawning, and egg hatching. The ORF in the SjZFP1 cDNA was 1017 bp in length and was predicted to encode a 338-aa protein with a molecular mass of approximately 38.5 kDa and theoretical isoelectric point (pI) of 7.08. Several conserved regions, including a B-box-type zinc-binding domain, two bipartite nuclear localization signal domains, a paired amphipathic helix repeat, and overlapping RING and PHD finger domains, were identified in the predicted amino acid sequence of SjZFP1. Using real-time PCR, we showed that the SjZFP1 mRNA was expressed across all of the developmental stages of the parasite and that the level of transcription was highest in the cercariae, eggs, schistosomula, and mature adult worms. The level of SjZFP1 mRNA expression in cultured schistosomula treated with one of two SjZFP1-specific small interfering RNAs (siRNAs; AY770 and AY546) was reduced by over 80 %, compared with that in the controls. In RNAi experiments in BALB/c mice, the level of SjZFP1 mRNA increased significantly when the mice were treated with the same SjZFP1-specific siRNAs during the early stages of infection. By contrast, the level of SjZFP1 mRNA decreased significantly when the mice were treated with the SjZFP1-specific siRNAs during the middle to late stages of infection. In four independent experiments, fewer worms were recovered from mice treated with the SjZFP1-specific siRNAs, compared with the number of worms recovered from the control mice. Both the average number and hatching rates of liver eggs recovered from mice treated with the SjZFP1-specific siRNAs during the middle to late stages of infection were significantly lower than those of the liver eggs recovered from the control mice. Our results suggest that the SjZFP1 gene might be important for parasite development, spawning in the vertebrate host, and egg hatching.

[Cloning, expression and immuno-protection analysis of a gene encoding troponin T of Schistosoma japonicum (SjTnT)].

OBJECTIVE: To clone cDNA encoding troponin T of Schistosoma japonicum (SjTnT), and evaluate the protective efficacy induced by recombinant SjTnT in BALB/c mice against S. japonicum challenge infection. METHODS: The SjTnT gene was amplified from 28-day-schistosome cDNAs by PCR and then subcloned into pET28a(+). The recombinant SjTnT protein (rSjTnT) was expressed in Escherichia coli BL21 (DE3) cells. The serum specific to rSjTnT was prepared by immunized BALB/c mice with the recombinant antigen, and the immunogenicity of rSjTnT was detected by Western blotting and ELISA. The immuno-protective efficacy induced by rSjTnT in BALB/c mice was evaluated according to the reduction in worm and egg counts. RESULTS: The cDNA encoding SjTnT was successfully cloned and expressed in E. coli. Western blotting showed that rSjTnT had a good immunogenicity. The high level of specific IgG antibodies was detected, and 33.89% worm reduction and 43.94% liver egg reduction were obtained in mice vaccinated with rSjTnT combined with Seppic 206 adjuvant compared with those in the adjuvant control group. CONCLUSIONS: rSjTnT could induce partial immuno-protection against S. japonicum infection in BALB/c mice. This study provided a basic for understanding the biological function of SjTnT.

[Distribution characteristics of deposited eggs and pathological changes in viscera of New Zealand white rabbits infected with Schistosoma japonicum at different time].

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.

Molecular characterization and identification of Th1 epitopes of a Schistosoma japonicum protein similar to prosaposin.

The tegument of schistosomula contains T cell antigens that might simulate the protective mechanisms of the radiation-attenuated vaccine in a mouse model of schistosomiasis. Immune mechanisms mediated by the CD4+ Th1 response are important in the RAV model. To rapidly identify Th1 epitopes in molecules from the Schistosoma japonicum schistosomula tegument, this study analyzed S. japonicum proteomics data. Preliminary experiments identified a protein similar to prosaposin (SjPSAP) from the tegument of schistosomula. We confirmed that SjPSAP was present in the tegument of the parasite using an indirect immunofluorescence assay. We then identified Th cell epitopes in SjPSAP using in silico prediction combined with experimental validation. From the SjPSAP sequence, we used several algorithms to predict 11 promiscuous Th cell epitopes that might bind to both murine and human MHC class II molecules. To validate the in silico predictions, proliferation and cytokine production profiles of spleen lymphocytes from BALB/c mice immunized with the 11 predicted peptides were measured in vitro using a modified methyl thiazolyl tetrazolium assay and flow cytometry. The results showed that 4 of the 11 predicted peptides induced a recall CD4+ Th1 response in vitro. We measured direct binding of the four peptides predicted to induce a response to antigen-presenting cells from BALB/c mice using a fluorometric method and found that the peptides bound to both I-Ad and I-Ed mouse molecules. These results demonstrated that potentially protective Th1-type epitopes in SjPSAP molecules could be identified rapidly by combining in silico prediction with experimental validation. This strategy could be a fast method for identifying Th1 epitopes in a schistosoma antigen with features such as large size or poor expression of recombinant antigens.

Seasonal dynamics of Schistosoma japonicum infection in buffaloes in the Poyang Lake region and suggestions on local treatment schemes.

Schistosomiasis japonica remains a major public health problem and the Poyang Lake region in Jiangxi province is one of the worst affected endemic areas. Buffaloes play a major role in the transmission of Schistosoma japonicum to humans. The aim of the present study was to increase understanding of the epidemic characteristics of schistosomiasis japonica in water buffaloes in the Poyang Lake region, after achieving the national mid-term goal, and to provide a basis for further interventions. The baseline prevalence in two villages in the Poyang Lake region in May 2010 was compared with respect to usage, sex and age in the total study population. Seasonal dynamics from May 2010 to May 2011 were observed in a natural village in the studied area. The baseline prevalence of infection in both villages (Caohui and Gaozhou) was 4.94% in May 2010. The prevalence in buffalo younger than 12 months was 12.82% in Caohui and 15.11% in Gaozhou, which was significantly higher than that found in those aged 13-24 months and older than 24 months. Of the 28 infected buffaloes, 82.14% (23) were younger than 12 months. The flow of seasonal dynamics showed that S. japonicum infection buffaloes were found from May to July and from November to January of the following year. This survey suggested that it is necessary to conduct two mass treatments (especially for young animals) in late March or early April and November, with an additional treatment of positive animals in July or June.

[Cloning, expression and immune function analysis of a gene encoding inositol monophosphate in Schistosoma japonicum].

OBJECTIVE: To clone and express a full-length cDNA encoding inositol monophosphate of Schistosoma japonicum (SjIM), and to access its immunoprotection in BALB/c mice for schistosomisis. METHODS: A full-length cDNA encoding the S. japonicum inositol monophosphate was isolated from 42 d schistosomes cDNAs. The expression profiles in different developmental stages were detected by real-time quantitative RT-PCR. The open reading frame (ORF) was subcloned into a pET28a(+) vector and transformed into BL21 and the recombinant protein was induced by IPTG. The immune characters of the purified recombinant protein were analyzed by Western blotting and immunoprotection in BALB/c mice. RESULTS: Bioinformatics analysis indicated that SjIM had an ORF of 834 base pairs that encoded 278 amino acids. Real-time quantitative RT-PCR analysis revealed that SjIM was upregulated in 35-day-old schistosomes, while the expression level in females was higher than that in male worms in 42nd day. Western blotting showed that the recombinant SjIM was immunogenic. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjIM could induce 48.76% and 41.29% reductions in the numbers of worms and eggs in the liver, respectively. CONCLUSIONS: The gene of SjIM is obtained from schistosomes cDNAs and the recombinant SjIM protein is induced successfully in E. coli. These aforementioned results demonstrate that the recombinant SjIM cand induce partial protection against schistosomiasis in BALB/c mice.

[Identification and preliminary analysis of a novel full-length cDNA encoding retinoid X receptor 2 from Schistosoma japonicum].

OBJECTIVE: To clone and preliminarily analyze the full-length cDNA encoding retinoid X receptor 2 (RXR2) from Schistosoma japonicum. METHODS: The rapid amplification cDNA ends (RACE)was applied to get a full-length cDNA encoding retinoid X receptor 2 from S. japonicum (SjRXR2). The transcription of SjRXR2 was detected by real-time PCR. By bioinformatical technology, the gene structure was analyzed and the antibody epitope was predicted. The polyclonal antibodies were raised in mice immunized with the synthesis peptide. Western blot was applied to detect its expression in the worm. RESULTS: The full-length cDNA of SjRXR2 was 5 960 bp and contained an open reading frame encoding a 1 435 amino acid which had a predicted molecular weight 159 kDa. Bioinformatical analysis indicated that SjRXR2 had a highly conserved DNA binding domain (DBD) and a moderate conserved ligand binding domain (LBD). The relative mRNA (s) of SjRXR2 with higher expressions at Day 21 and 42 were evaluated in five different S. japonicum developmental stages. The Western blot analysis showed that polyclonal antibodies were able to specifically recognize the protein with molecular around 150 kDa from the extract of S. japonicum. CONCLUSION: A full-length cDNA encoding retinoid X receptor 2 (RXR2) from S. japonicum is obtained which provides preliminary information for further investigation of SjRXR2 functions in S. japonicum.

[Screening and verification on characteristic differentially expressed genes of Schistosoma japonicum from three reservoir hosts].

OBJECTIVE: To get the characteristic differentially expressed genes of Schistosoma japonicum from three important reservoir hosts: yellow cattle, water buffalo and goat, so as to find the genetic markers to identify the various sources of the parasite reservoir hosts. METHODS: The 49 d worms were collected from artificially infected animals, and the total RNA(s) of worms were extracted and reverse-transcripted to cDNA, and then hybridized with custom-built microarray to screen characteristic differentially expressed genes of every host, and the microarray results were validated by the real-time PCR method. RESULTS: From results of microarray, we got 3 characteristic differentially expressed genes of S. japonicum from yellow cattle, 4 from water buffalo and 7 from goat. We verified schistosome samples from three reservoir hosts in another experiment, the results showed that 2 in yellow cattle, 3 in water buffalo, and 5 in goat were verified to be consistent with microarray results. CONCLUSIONS: The ten characteristic differentially expressed genes of S. japonicum from three reservoir hosts screened by microarray might be used as genetic markers to identify the various sources of reservoir hosts for S. japonicum.

[Function analysis of Sj34.9 gene based on RNAi and microarray].

OBJECTIVE: To analyze the function of Sj34.9 gene, so as to provide the reference for future studies. METHODS: The Sj34.9 gene was knocked down in Schistosoma japonicum by RNA interference (RNAi), and the microarray was used to analyze the genes'expression of S. japonicum after Sj34.9 knocked down. RESULTS: A total of 378 genes expressed differently including 202 up-regulated genes and 176 down-regulated genes. The pathway analysis indicated that the genes expressed differently were mainly related to organelles, metabolism and signal transduction. The gene ontology category analysis showed that most of these genes might be involved in binding, membrane fomulation and cellular process. CONCLUSION: The gene Sj34.9 might play important roles in the process of growth, development, reproduction and metabolism of S. japonicum.

Identification of Th1 epitopes within molecules from the lung-stage schistosomulum of Schistosoma japonicum by combining prediction analysis of the transcriptome with experimental validation.

The lung-stage schistosomulum has been regarded as the main target of protective immunity induced by radiation-attenuated vaccines (RAV) in the mouse model of schistosomiasis, and immune mechanisms mediated by the CD4+ Th1 response play a major role in the RAV model. To identify Th1 epitopes rapidly within molecules from the lung schistosomulum of Schistosoma japonicum, in the present study we analyzed transcriptome data from normal and radiation-attenuated lung schistosomula of S. japonicum and Schistosoma mansoni. We selected six genes with high levels of expression of their transcripts as sample sequences from the lung schistosomula. From these six sequences, by using different algorithms, we predicted six promiscuous Th cell epitopes that are capable of binding to both murine and human MHC class II molecules. To validate our in silico prediction experimentally, first, the gene expressions of the six sequences in day 3 lung-stage schistosomula were assessed using reverse-transcription PCR (polymerase chain reaction) analysis. The result showed that all six sequences predicted can be expressed in normal day 3 schistosomula. Second, we measured the direct binding of the four peptides predicted above to APCs (Antigen Presenting Cells) from the BALB/c mouse strain using a fluorometric method, and found that the four peptides could bind to both I-Ad and I-Ed molecules of the mice. Finally, the proliferation and profiles of cytokine production by spleen lymphocytes from the BALB/c mice immunized with the six predicted peptides were detected in vitro using modified MTT (Methyl Thiazolyl Tetrazolium), and flow cytometry methods, respectively. The results showed that three of the six predicted peptides could induce a recall CD4+ Th1 response in vitro. These results demonstrate that potential Th1-type epitopes can be identified rapidly by a combination of in silico analysis of transcriptomes of lung-stage schistosomula with experimental validation.

[Longitudinal observation of epidemic dynamics of schistosomiasis in bovine in two mountainous endemic regions].

OBJECTIVE: To understand the endemic situation dynamics of schistosomiasis in domestic animals (mainly bovine) in mountainous endemic regions, so as to provide the reference for evaluating the control effect and improving control strategy. METHODS: Two representative pilots (Renmei and Dacang) in mountainous schistosomiasis endemic regions were selected for survey. The schistosome infection status of bovine was investigated by the miracidium hatching method, the pasture of bovine were investigated by home visiting, and the distributions of wild feces and Oncomelania snails, and the snail schistosome infection status were also investigated in April and September every year. RESULTS: The schistosome infection rates of bovine reduced by 98.4% and 93.8% in two pilots in 2007 compared with those in 1993, and the infection intensities also showed a decline trend. The infection rate of wild faces was 0 in Renmei pilot since 1995, while in Dacang pilot, the infection rate of wild feces fluctuated in 2007, and the intensities of living snails and infected snails showed a declined trend. CONCLUSIONS: Due to the special natural environment of mountainous endemic regions, there is a dot-like or band-like distribution of endemic areas. The strengthening of schistosomiasis examination and chemotherapy will rapidly reduce endemic situation. However, to completely interrupt the transmission of schistosomiasis, we should emphasize environmental modification and domestic animal management.

[Screening interaction protein of Schistosoma japonicum gynecophoral canal protein by phage display].

OBJECTIVE: To search the interaction protein of Schistosoma japonicum gynecophoral canal protein (SjGCP). METHODS: The recombinant rSjGCP was used as a target to search the T7 phage display cDNA library from 44-day adult Schistosoma japonicum. RESULTS: A total of 70 ESTs were obtained. The bioinformatics analyses to the screening results revealed that 5 interaction proteins or peptides of SjGCP were found. CONCLUSION; Interaction proteins or peptides of SjGCP are successfully obtained through screening the T7 phage display library.

In silico prediction of binding of promiscuous peptides to multiple MHC class-II molecules identifies the Th1 cell epitopes from secreted and transmembrane proteins of Schistosoma japonicum in BALB/c mice.

It has not so far been possible to identify rapidly and effectively the anti-schistosomiasis Th cell epitopes that are capable of simulating IFN-γ (Interferon-gamma)-mediated Th1-type protective immunity in response to radiation-attenuated schistosome cercaria. With the advance of the omics studies of schistosomes, an approach that used reverse vaccinology probably resolved the above problems. In this "proof-of-principle" study, first, we selected 31 secreted or transmembrane protein sequences sampled from sequences of the transcriptome of Schistosoma japonicum, and analyzed characteristics of these proteins by using conventional bioinformatics tools. Second, putative promiscuous Th cell epitopes within these proteins were predicted using three to four different immuno-informatics algorithms for the prediction of MHC (Major Histocompatibility Complex) class-II binding peptides. We predicted using these in silico approaches promiscuous Th cell epitopes that are capable of binding to both murine and human MHC class-II molecules. To validate our in silico prediction experimentally, BALB/c mice were immunized with the five predicted peptides, and the proliferative responses and cytokine production of lymphocytes from the immunized BALB/c mice were assessed in vitro by modified MTT (Methyl Thiazolyl Tetrazolium), ELISA (Enzyme-linked Immunosorbent Assay) and flow cytometry methods. The results showed that two of the five predicted peptides could induce a Th1-type response in vitro. These results suggest that promiscuous Th1 cell epitopes from secreted or transmembrane proteins of S. japonicum can be identified using a strategy of reverse vaccinology.