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.

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.

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.

RNAi silencing of calcium-regulated heat-stable protein of 24 kDa in Schistosoma japonicum affects parasite growth.

The calcium-regulated heat-stable protein of 24 kDa (CRHSP-24) is a major calcineurin phosphoprotein that functions in multiple signal transduction pathways in cell metabolism. Schistosomes are multicellular parasites that infect 200 million people worldwide, even though treatment has been available for two decades. To determine the function of schistosome CRHSP-24 (SjCRHSP-24), we successfully knocked down SjCRHSP-24 in Schistosoma japonicum by RNA interference (RNAi). By establishing controls for measuring off-target RNAi effects, we found that different double-stranded (dsRNA) sequences had different levels of effectiveness. While all tested dsRNAs reduced CRHSP-24 transcript levels, the S2 dsRNA consistently reduced CRHSP-24 expression to >95% of the control. Knockdown of the SjCRHSP-24 gene significantly affected the morphology and vitality of S. japonicum.

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.

Wnt4, the first member of the Wnt family identified in Schistosoma japonicum, regulates worm development by the canonical pathway.

The Wnt signaling pathway is an evolutionarily conserved signal transduction pathway used extensively during animal development. We aim, by increasing our understanding of the Wnt signaling pathway, to find a key gene or protein present in schistosomes that can be developed into vaccine candidate or drug target. We therefore isolated the Wnt4 gene from Schistosoma japonicum. Wnt4 encodes a putative protein of 558 amino acids which contains the conserved functional domain of the Wnt gene family. We suppressed the expression of Wnt4 mRNA in 10-day schistosomulae by RNA interference. Quantitative PCR analysis showed that Wnt4 displayed a 73% reduction in the transcript level. And GSK-3beta and beta-catenin, which are involved in Wnt canonical pathway, showed a 45% and 39% reduction in mRNA levels, respectively. PLC, CaMKII, DVL, and JNK, which are involved in Wnt non-canonical pathway, showed no reduction. These results suggest that the Wnt4 signal protein in S. japonicum regulates downstream genes by a canonical pathway. Wnt4 is the first member of the Wnt family to be identified in S. japonicum. An increased understanding of the Wnt signal transduction pathway will allow us to elucidate further the molecular mechanism of development in schistosomes.

[Evaluation on the immuno-protective efficacy of the recombinant antigen SjPGAM-SjEnol against Schistosoma japonicum in mice].

OBJECTIVE: To construct and express the recombinant plasmid pET32a-SjPGAM-SjEnol and evaluate its immuno-protective efficacy against the infection of Schistosoma japonicum in mice. METHODS: The peptides of SjPGAM and SjEnol containing the multivalent epitopes with higher binding capacity of human MHC II and mouse H2-dII but low homology with the host were analyzed and screened through bioinformatics. The corresponding nucleotide sequence of selected epitopes was spliced and the recombinant plasmid pET32a-SjPGAM-SjEnol was constructed and expressed in Escherichia coli BL21 cells. The antigenicity of the recombinant protein was detected by Western blotting and the protective effect induced with the recombinant was evaluated in mice. 55 BALB/c mice were randomly divided into 5 groups each with 11. Mice from groups A, B and C were injected with a mixture of recombinant protein (27 microg) pET32a-SjPGAM-SjEnol (A), pETL28a-SjPGAM (B) and pET28a-SjEnol (C) respectively together with 206 adjuvant, mice from groups D and E received adjuvant or PBS only, all injected for three times at two-week intervals. Mice were then challenged with 40 +/- 2 cercariae of S. japonicum at two weeks after the last vaccination, and sacrificed for perfusion by 6 weeks post infection. Adult worms were collected, the number of eggs in a gram of liver tissue was counted, and the rates of worm reduction and egg reduction were calculated. Serum samples were collected before vaccination, every one week after each inoculation and before sacrifice, and specific IgG was detected by ELISA. RESULTS: The sequences encoding the 96-147 aa of SjPGAM and 233-312 aa of SjEonl were chosen for constructing the recombinant plasmid, a cDNA fragment with the length of 447 bp was amplified by PCR. The recombinant plasmid was expressed in E. coli with a molecular weight of Mr 33,000. Western blotting revealed that the fusion protein was recognized by the rabbit serum specific to SjSWAP, and showed an adequate antigenicity. Vaccination experiment showed that when compared with those of the blank control, the worm reduction rate in group A was 39.7%, significantly higher than that of groups B (18.5%) and C (14.7%) (P < 0.05). The liver egg reduction rate in group A was 64.9%, also higher than that of groups B (47.5%, P < 0.05) and C (30.5%, P < 0.01). ELISA showed that the serum specific IgG in group A (2.372 +/- 0.268) was much higher than that of groups D (0.490 +/- 0.138) (P < 0.01 and E (0.220 +/- 0.088) (P < 0.01). CONCLUSION: The recombinant plasmid pET32a-SjPGAM-SjEnol has been constructed, and recombinant protein pET32a-SjPGAM-SjEnol induces higher immune-protection against S. japonicum than that of SjPGAM and SjEonl.

[Immune protection of tegument protein rSj29 against Schistosoma japonicum in mice].

OBJECTIVE: To clone, express and characterize a tegument protein gene of Schistosoma japonicum (Sj29) , and investigate the immune protection of the recombinant protein against S. japonicum in mice. METHODS: The gene coding for Sj29 protein was amplified by PCR, and the sequence was analyzed by bioinformatics tools. Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced the recombinant with IPTG. The recombinant protein (rSj29) was purified by His-binding-resin affinity chromatography and characterized by Western blotting. Three groups each with 10 BALB/c mice were immunized subcutaneously three times (two weeks interval) respectively with 100 microl recombinant rSj29 (0.1 mg/ml) , adjuvant or PBS. At the 15th day after the final inoculation, each mouse was challenged by 40 +/- 2 cercariae of S. japonicum. At the 53rd day after infection, the mice were sacrificed to obtain the number of adult worms, number of eggs in liver and feces. Serum samples were collected at pre-immunization and certain time after immunization, and were analyzed for IgG by ELISA. The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique. mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR. RESULTS: A 576 bp Sj29 gene fragment was obtained. The recombinant protein rSj29 with Mr 22,900 was expressed in the form of inclusion body. The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice. The number of adult worms (15.4 +/- 5.9), number of hepatic eggs (40,143.3 +/- 2,995.9) and number of fecal eggs (3,803.9 +/- 110.9) in recombinant protein group were significantly higher than those of PBS control group (20 +/- 3.4, 49,318.1 +/- 6,648.3, 5,238.1 +/- 303.5, respectively) (P < 0.05) . There was a high level of specific IgG against rSj29 (maximum dilution 1:32000) in recombinant protein group. Immunohistochemical analysis showed the Sj29 protein expressed on the surface of different stages of S. japonicum. mRNA level of Sj29 was the highest at the 32nd day post-infection. CONCLUSION: The recombinant protein rSj29 induces certain degree of protective immunity in mice.

[Construction and expression of recombinant baculovirus with Schistosoma japonicum SjPP gene].

OBJECTIVE: To express the soluble recombinant Schistosoma japonicum SjPP proteins in TN5B1-4 cells. METHODS: The total RNA was extracted from adult worms of Schistosoma japonicum. The whole coding sequence of SjPP gene was synthesized by RT-PCR and cloned into donor plasmid. The recombinant donor pFastBac-SjPP was transformed into E.coli DH10Bac forming Bacmid-SjPP which was transfected into insect cell with cational lipofectin. The fusion protein SjPP was analyzed with SDS-PAGE and Western blotting. RESULTS: The infective recombinant baculovirus Bacmid-SjPP was obtained and SjPP protein was expressed in insect cells. CONCLUSION: The recombinant protein SjPP has been expressed in insect TN5B1-4 cells with proper antigenicity.

[Effect of IgG3 antibody purified from sera of Microtus fortis against Schistosoma japonicum].

IgG3 antibody reaction to soluble antigens prepared from schistosomula (SSA), adult worms (SAWA) and eggs (SEA) in laboratory-bred Microtus fortis (Mf), BALB/c mice and Kunming (Km) mice challenged by cercariae of Schistosoma japonicum was detected by indirect ELISA. The effect of purified IgG3 antibody on in vitro killing schistosomula and protecting mice from infection of S. japonicum was evaluated. The IgG3 antibody level in Mf against SSA and SAWA increased significantly by 79.6 percent and 49.6 percent after the fourth week of challenge infection, but no significant increase in BALB/c mice. Purified IgG3 antibody from laboratory-bred Mf and wild Mf effectively killed schistosomula, and that of the wild Mf induced higher worm-reduction rate. The death rate of schistosomula due to IgG3 antibody purified from sera of laboratory-bred Mf and wild Mf was 2.35 and 5.88 times as high as that of Km mice respectively. The results suggest that IgG3 antibody from Microtus fortis may play an important role in immunity against S. japonicum.

[Application of reverse vaccinology in Schistosoma vaccine development: advances and prospects].

OBJECTIVE: Current advances in reverse vaccinology based on the principle of "sequence-structure-function" and such integrated platform technologies as immunoinformatics, computer-aid design, and various high-throughput omics (including genomics, transcriptomics and proteomics) may pave a new way for the discovery of candidate vaccine molecules against schistosomiasis. Both theoretical prediction and experimental approaches conventionally used in the field of reverse vaccinology are briefly introduced in this review; and the applications of these approaches to screening and confirming candidate Schistosoma vaccine molecules are also summarized. Furthermore, potential research prospects of the application of reverse vaccinology to Schistosoma vaccine development are discussed by simulating immune effect mechanisms of immunization with radiation-attenuated cercaria vaccine in animal hosts and naturally acquired immunity in human population.

[Construction of phage display cDNA library from adult worms of Schistosoma japonicum].

OBJECTIVE: To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. METHODS: Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. RESULT: Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library. CONCLUSION: The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

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.

[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.

[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.

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.

Cloning and Expression of Actin Gene of Schistosoma japonicum Chinese Strain.

A 1 131 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with primers designed according to published SmAct2 encoding Schistosoma mansoni actin. Sequence analysis indicated that this fragment, with 92% homology to SmAct2, was a complete open reading fragment (ORF) of actin gene of Schistosoma japonicum (Chinese strain). This gene was cloned into the expression vector pET28a( ) and subsequently expressed in Escerichia coli. SDS-PAGE revealed that the molecular weight of this expressed product was 47 kD. Western blotting showed that the recombinant protein had good reactivity with the rabbit serum immunized with Sj worm antigen, indicating that this gene encode actin of Schistosoma japonicum(Chinese strain).

Cloning of Schistosoma japonicum Chinese Strain TPI Gene and Characterization of Its Expression Product in Escherichia coli.

Triose-phosphate isomerase is an important candidate for schistosoma antigens. An 800 bp DNA fragment was amplified by RT-PCR from adult Schistosoma japonicum mRNA. Sequence analysis revealed that this fragment contained S. japonicum (Chinese strain) triose-phosphate isomerase gene. Then this gene was cloned into the expression vector pGEX-4T and subsequently expressed in E. coli. The recombinant GST-fusion protein was purified by glutathione agarose affinity chromatography. Its molecular weight was determined to be 54 kD. The yield of expression was around 30 mg/L E. coli culture. Western blotting showed that the recombinant protein had good antigenicity which could be helpful for the making of anti-S. japonicum multi-valent recombinant vaccine.

[Cloning, expressing and functional analysis of SjMF4, a novel Schistosoma japonicum gene].

Based on the phenomenon of the natural anti-schistosomiasis in Microtus fortis, the sera from normal Microtus fortis were employed to immunoscreen the cDNA library of adult Schistosoma japonicum (Chinese strain), two positive clones were obtained, RACE technique was further applied to amplify one of the clones, and a cDNA fragment with an ORF was identified. Sequencing revealed that it was a novel gene of Schistosoma japonicum, and it was named SjMF4 (Schistosoma japonicum Microtus fortis 4). Then the structure and functional motifs of SjMF4 were analysed. The gene was subcloned into pET-28a(+) vector; the recombinant protein showed good antigenicity in Western blotting. The gene was further subcloned into eukaryotic expression vector pcDNA3 to construct the DNA vaccine containing SjMF4. Immune experiments in mice showed significant protection that the recombinant plasmid did induce 28.64%+/-3.82% worm reduction and 21.73%+/-3.98% egg reduction than controls against the Schistosoma japonicum cercaria challenge.