[Biological identification on sub-cultivation cells of Schistosoma japonicum adult worms in vitro].

Cultivation of cells from 30-day old Schistosoma japonicum (S.j) adult worms showed that the growth features of the cells were semi-floating and accumulative. The survival rate of the primary cells, passage cells prior to the 5th generation and recovered cells was all up to 90%. Phases of cell division were observed during cultivation. Chromosome karyotype of the 5th generation cells possessed diploid feature of the blood-flukes (2n=8 in number). Ultrastructure of the 5th generation cells showed that four types of cells in normal morphology and three types of cells in abnormal morphology were both viewed. It is suggested that some of the cells from S.j adult worms were subcultured successfuly in the 1640-40 defined medium.

[Study on the relationship of immune status with severity of schistosomiasis japonica in fishermen in the Dongting Lake].

OBJECTIVE: To investigate the relationship of the immune status and the intensity of infection or the severity of the hepatosplenic pathology among fishermen with schistosomiasis japonica in the Dongting Lake region. METHODS: Inquiring and physical examination (IPE), stool examination, B-ultrasonography of the liver and spleen, flow cytometry, turbidimetry and ELISA were undertaken to acquire or determine the intensity of infection (EPG in stool), pathological change in the liver and spleen and the level of cellular and humoral immunity. Data were analyzed with SPSS 10.0 statistics software. RESULTS: Compared with subjects from non-endemic area, the CD4+ T cells and the CD4+/CD8+ ratio in fishing population in the endemic area significantly decreased. The decrease of the CD4+/CD8+ ratio was more significant among population with positive stool exam and with the increase of EPG and/or severity of pathological change in the liver and spleen. Contrarily, the level of the total IgM and the anti-SEA IgG in serum from fishing population in the endemic area was significantly higher than those from non-endemic area. High level serum antibodies in those stool positives were remarkable with the increase of EPG and/or the severity of hepatosplenic pathological change. The total IgA increased considerably in the subjects with significant pathological change of the liver and spleen. A high total IgG was only detected in those stool positives. CONCLUSION: The immune status in fishermen with schistosomiasis in the Dongting Lake showed a suppressed cellular immunity and a hyper functioning humoral immune response. The imbalance of the immunity was related to the increase of the intensity of infection and the progress of the hepatosplenic pathology.

[Boost effect of recombinant IL-4 on protection of Schistosoma japonicum cathepsin B DNA vaccine in mice against the parasite].

OBJECTIVE: To investigate the enhancement effect of IL-4 expression plasmid on cathepsin B DNA vaccine of Schistosoma japonicum (Sj) in mice. METHODS: The recombinant IL-4 plasmid constructed by cloning PCR amplified product of murine IL-4 gene into eukaryotic expression vector pcDNA3.1 was co-injected intramuscularly with Sj cathepsin B expression plasmid DNA to mice as the test group. The other three groups of mice were set up as control including IL-4 expression plasmid, Sj cathepsin B expression plasmid and two vacant vector plasmids. The expression of IL-4 and cathepsin B was visualized by immunohistochemistry. Challenge infection in mice was carried out 3 weeks after the last vaccination and immune protection was assessed by worm and egg reduction rates. RESULTS: The recombinant mIL-4 plasmid and cathepsin B DNA vaccine were expressed in muscular cells of the vaccinated mice. Immunization with cathepsin B DNA plus recombinant mIL-4 plasmid yielded a 43.2 % of worm reduction rate and a 76.6% of egg reduction rate, showing a significant difference (P<0.01, P<0.05) compared with that of cathepsin B DNA vaccine alone. CONCLUSION: As an adjuvant, IL-4 DNA can improve the protective effect of cathepsin B DNA vaccine in mice against S. japonicum infection.

[Analysis on morbidity and chemotherapy effects of Schistosoma japonicum infection in fishermen on Dongting Lake].

OBJECTIVE: To clarify and evaluate the morbidity of schistosome infection and the effectiveness of chemotherapy among fishermen on East Dongting Lake. METHODS: Information on water-contact, history of infection and of praziquantel (PZQ) treatment among fishermen was collected. Kato-Katz method and miracidium hatching test were applied to detect the pathogens in stool specimen. Serum antibodies against soluble egg antigen (SEA) were detected with ELISA and IHA. B-ultrasonic examination was used to determine the pathological changes of liver and spleen. Chemotherapy [PZQ 40 mg/(kg x d)] was given to the fishermen followed by a re-examination after a transmission season. RESULTS: The first investigation (six months before chemotherapy) showed that among 268 people inquired, 90.7% were frequently or intermittently contacting water, 24.0% were treated with PZQ each year, 39.4% had never been treated in the recent five years. Stool positive rate was 68.1% (111/163) and the geometric mean eggs per gram feces (EPG) were 48.77. Males had a higher infection rate (76.0%) and intensity (62.97 EPG) compared with that of females (58.7% infection rate and 30.42 EPG). The highest positive rate (83.3%) was in the age group of 11 to 20 years old. The prevalence of those who frequently or intermittently contacted water and were never treated before was 76.3% (106/139) and 79.7% (51/64), respectively. Serological positive rate was 88.0% (IHA) or 78.7% (ELISA). B-ultrasound revealed 77.4% (82/106) of the fishermen showing pathological changes in liver and/or spleen due to schistosomiasis. 37.7% of the patients showed II-III stage liver fibrosis (male: 53.0%, female: 15%), 58.5% hepatomegaly and 19.8% splenomegaly. The second investigation (six months after chemotherapy with PZQ) showed a stool positive rate of 35.4% and an average EPG 36.13 in the treatment group which were considerably lower than 56.5% infection rate and 68.47 EPG in the group without treatment. In 39 patients treated, the reversion rate from egg positive to negative was 48.7%, pathological change in liver and spleen declined by 40.6%. CONCLUSION: The prevalence and morbidity of schistosomiasis in fishermen on Dongting Lake were high due to frequent exposure to the affected water, and chemotherapy can effectively reduce the prevalence, the intensity of infection and morbidity of the fishermen.

[Mucosal immunization of recombinant Schistosoma japonicum ferritin].

OBJECTIVE: To subclone and express the gene encoding Schistosoma japonicum ferritin (SjFer) and study its immune protection against challenge infection in mice vaccinated intranasally. METHODS: The SjFer gene was amplified by PCR, and subcloned into the N-terminal of intein 2 of the pTWIN1 vector. The positive recombinant was screened by PCR, restriction enzyme digestion and sequence analysis. The positive recombinant was transformed into E. coli ER2566. The soluble recombinant fusion protein (rSjFer-intein 2) was expressed in E. coli by induction of low IPTG concentration under low temperature, and analyzed by SDS-PAGE and Western blotting. Mice were immunized intranasally with rSjFer, using chitosan as adjuvant. Two weeks after the third vaccination, challenge infection with S. japonicum cercariae was carried out. Worms and eggs collected from the livers of mice were counted at 42 days after the challenge. Levels of specific antibodies were detected by ELISA before infection. RESULTS: SjFer was successfully subcloned into pTWIN1 vector and expressed in E. coli. In mice vaccinated intranasally with rSjFer and adjuvant chitosan, the worm reduction rate was 35.51% and the reduction rate of eggs per gram liver tissue (LEPG) was 52.17%. As compared with the control groups, levels of IgG, IgA in sera and SIgA in saliva increased significantly. CONCLUSION: The expressed rSjFer can induce partial protective immunity against S. japonicum infection in mice when they were vaccinated intranasally, with chitosan as adjuvant.

Schistosoma japonicum: isolation and identification of peptides mimicking ferritin epitopes from phage display library.

In an attempt to isolate and identify the antigenic epitopes on ferritin of Schistosoma japonicum (SjFer) and to test their protective potentiality against Schistosoma japonicum (S.j), polyclonal antisera against SjFer was prepared to screen a 12-mer random peptide library. Three rounds of biopanning were performed and resulted in an enrichment. Six peptides selected randomly from the third elute were all found to be positive by evaluating the binding to anti-SjFer sera by ELISA and Western blotting. Three amino acid sequences were deduced from the six phage clones by sequencing. When they were used to vaccinate mice, the three peptides could induce significant reduction in adult worms (26.7%, 20.4%, and 25.9%) as well as in liver eggs per gram (LEPG) (40.0%, 38.2%, and 40.8%). This result showed that three mimotopes on SjFer were obtained and they could induce significant protective efficacy against S.j.

Molecular cloning, expression and vaccination of a novel gene Sj-MA of Schistosoma japonicum.

In order to obtain new gene and to develop the new vaccine candidate of immunoprotection against Schistosoma japonicum (Sj), Sj adult worm cDNA library was screened with anti-sera to soluble male adult worm antigen and resulted in the discovery of a novel gene designated as Sj-MA. Sequence analysis showed that Sj-MA as a complete cDNA contains one open reading frame. It was deduced to contain 249 amino acid residues and encode a 28.8 kD soluble protein with plenty of phosphorylation sites, supposing its action in signal transduction. Furthermore, Sj-MA cDNA was cloned into a prokaryotic expression vector pGEX-5X to construct the recombinant plasmid which was transformed and highly expressed in E. coli as a 54.8 kD glutathione-S-transferase (GST) fusion protein. The fusion protein rSj-MA/GST could be recognized with both anti-male adult worm sera and anti-GST sera in Western blotting. Mice vaccinated with the fusion protein revealed significant worm reduction rate of 34.29% (P<0.001), compared with the control groups. Taken together, the novel gene Sj-MA can be expressed in E. coli as a fusion protein that can elicit immunity against Schistosoma japonicum, suggesting its potential as a new vaccine candidate.

[Studies on fractionation and protective immunity of the membrane antigen from hepato-portal juveniles of Schistosoma japonicum].

OBJECTIVE: To explore the immunological characteristics of the membrane antigen from hepato-portal juveniles of Schistosoma japonicum and its protective immunity against S. japonicum (Sj) in mice. METHODS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and enzyme-linked immune electro-transfer blot(EITB) methods were used to recognize the membrane antigens from hepato-portal schistosomula (SjHmAg) by infected rabbit sera (IRS) and normal rabbit sera (NRS). Kunming mice were immunized subcutaneously three times(0, 2, 4 weeks) with SjHmAg. Each mouse was challenged with 40 +/- 1 cercariae. Six weeks later the mice were killed, worms and liver eggs were counted. RESULTS: 7 major protein bands appeared on SDS-PAGE. IRS mainly reacted specifically with SjHmAg of 23, 33 and 63 kDa. Compared with the control groups, the reduction rate of worms and eggs per gram liver in the experimental group were 16.2% and 54.4%, respectively. CONCLUSION: Different protein components from SjHmAg are obtained using SDS-PAGE, and the antigen can induce a protective immunity against Sj in mice.

[Protective effect against Schistosoma japonicum of recombinant fusion protein SjGST-32 in mice].

OBJECTIVE: To explore the optimal immune doses of recombinant antigen rSjGST-32, with QuilA as adjuvant. METHODS: BALB/c mice were immunized with doses of 50, 100 and 200 micrograms rSjGST-32/mouse plus 50 micrograms QuilA adjuvant. QuilA and PBS were used as controls. Levels of specific antibodies were detected by ELISA. The mice were challenged 4 weeks after last vaccination. Worms and eggs collected from the livers of mice were counted 45 days after challenge. RESULTS: As compared with the control groups, the worm reduction rate in the 50, 100 and 200 micrograms experiment groups was 38.1%, 47.8% and 48.8%, respectively. The reduction rate of liver eggs per gram (LEPG) was 39.1%, 53.5% and 53.6%, respectively, and the reduction rate of the liver eggs per female (LEPF) was 22.5%, 22.8% and 21.8%, respectively. The specific antibody titers in sera reached 1:51,200, 1:102,400 and 1:102,400, respectively before challenge. CONCLUSION: The results show that for vaccinating BALB/c mice, the optimal antigen dose is 100 micrograms of recombinant rSjGST-32 plus QuilA adjuvant.

[Intranasal or intragastric vaccination of mice with recombinant Schistosoma japonicum ferritin induces immune protection against challenge infection].

OBJECTIVE: To test the immune protection against challenge infection in mice vaccinated intranasally or intragastrically with recombinant Schistosoma japonicum (S.j) Ferritin (rSjFer). METHODS: Mice were divided into 8 groups each with 10 mice. They were immunized intragastically with rSjFer, CTB, rSjFer + CTB and intranasally with rSjFer, CTB and rSjFer + CTB respectively. PBS was used intragastically or intranasally as control groups. The mice were challenged with 40 +/- 1 S. j cercariae per mouse 2 wk after the third vaccinization. Forty-five days later, mice were killed and perfused, and the adult worms and eggs were counted. Serum and fecal samples were obtained before the first immunization and the challenge infection. IgA and IgG in sera and sIgA in feces were detected by ELISA. RESULTS: The worm reduction rate was 3.98%, 3.77%, 25.57% in the intragastric vaccination groups and 7.59%, 4.50%, 33.35% in the intranasal vaccination groups respectively. The egg reduction rate was 3.76%, 2.46%, 34.75% and 4.40%, 0.06%, 60.10% respectively. CONCLUSION: This study showed that a significant immune protection against Schistosoma japonicum infection was induced by mucosal (intranasal and intragastic) vaccination with rSjFer.

[Study on the subcloning, expression and immunoprotection with Schistosoma japonicum CAI gene].

OBJECTIVE: To subclone and express the new gene of Schistosoma japonicum (Sj) CAI and evaluate the immunoprotective effect of the recombinant molecule. METHODS: The cDNA of SjCAI gene was subcloned into expression vector pGEX-5X-3 to form recombinants which were then used to transform to E. coli strain ER 2566. Expression was induced by IPTG. The mice were vaccinated with the expressed protein and the immunoprotective effect was tested. RESULTS: Fusion protein of SjGST-CAI was highly expressed in E. coli as inclusion bodies. The worm reduction rate and the liver egg reduction rate in vaccination group of SjGST-CAI were 29.87% and 63.71%, respectively. CONCLUSION: SjCAI gene can be highly expressed in E. coli after subcloning into pGEX-5X-3 vector and the expressed fusion protein can induce immunoprotective effect against Sj in mice.

Cloning, expression and immunization of the new antigen gene Sj-Ts4 of Schistosoma japonicum.

In order to explore the molecular mechanism of high immune protection against schistosomes infection in animals infected with Trichinella spiralis, and to provide several cross-protective antigen genes for developing anti-schistosomiasis vaccine, a Schistosoma japonicum adult worm cDNA library was immunoscreened using sera taken from mice infected with Trichinella spiralis. Nine positive clones were obtained after 3 rounds of immunoscreening. Among them, Sj-Ts4 represents a novel gene of S. japonicum, which coding for a novel protein of 210 amino acids. The protein has a deduced molecular mass of 23 kD and isoelectric point of 7.72. Sj-Ts4 was expressed as a glutathione-S-transferase (GST) fusion protein by cloning into the prokaryotic expression vector pGEX-5X-3. The recombinant Sj-Ts4 protein (rSj-Ts4) was purified and could be recognized by sera of mice infected with S.japonicum. Vaccination of several groups of mice with rSj-Ts4 or rSj-Ts4 incorporated into Freund's complete adjuvant induced high titers of specific IgG antibodies. Two vaccination groups all obtained significant reduction in worm burden (31.36%, 36.80%, P<0.01), compared with the control groups.