In order to explore the available sero-diagnotic method for surveillance in basically controlled and controlled areas of schistosomiasis japonica, a 3 years longitudinal investigation of children 6~14 years old with COPT in 5 endemic areas was carried out after being basically controlled for 8~16 years. The results showed that the examination of children with COPT is one of the available serodiagnostic methods of schistosomiasis for surveillance. In all five areas the positive rate of children in this age group was less than 3%, and the circumoval precipitin rate among the positive cases was less than 5%. Therefore, it is indicated that the above mentioned two indexes can be used for sero-surveillance of schistosomiasis japonica.

A kind of colloidal blue dye(D-l)used as a staining reagent for immunoassay was first selected from the dyes produced in China in this study. The optimun condition for labelling the dye onto sheep antihuman IgG antibody was explored. The dye-labelled antibody could react and stain with relative antigens. The minimal concentration of human IgG protein could be detected with the labelled dye by Dot Double Antibody Sandwich Assay was 20-10ng/ ml. 61 of 62 sera samples of schistosomiasis japonica were positive,the positive rate was 98.4%. Just 1 out of 30 healthy people's sera sambles was positive,the false positive rate was 3.3%. All of 40 Fasciolopsiasis sera samples were negative. The results were similar to that of Dot-ELISA. The activity of antibody labelled with colloidal dye could be maintained for 1 week in room temperature and be presered in lyophilized condition for a longer period. The assay was less expensive,simple to conduct and required no special equipment. It suggested that the Dot Colloidal Dye Immunoassay(DIA)might have potential value for use in schisto-somiasis endemic areas.

Objective To evaluate the effect of DDIA on detecting cattle schistosomiasis. Methods The sera of schistosomiasis cattle, negative control cattle and fascioliasis cattle were detected with DDIA and compared with COPT. Results The sensitivity, specificity and across reaction of DDIA using 10 ?l of sample sera were 95.7%, 96.0% and 20.0% respectively and COPT were 72.9%, 100.0% and 0 respectively. Conclusions DDIA has high sensitivity and specificity for detecting cattle schistosomiasis with 10 ?l of sample serum, and the sensitivity is higher than that of COPT.

Objectives To express the miracidial antigen from eggs of Schistosoma japonicum (Chinese mainland strain) (SjMP10), and investigate the role of the miracidial antigen during the hepatic granuloma formation of schistosomiasis. Methods A pair of specific primers was designed and synthesized according to the nucleotide sequence of the open reading frame of the miracidial antigen gene. The open reading frame of the miracidial antigen gene was amplified, digested by restrictive enzyme(BamHI, SalI), and cloned directly into the expression plasmid pGEX-4T-3 to construct the recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21(DE3), and induced by IPTG to express the fusion protein of GST-SjMP10. The expressed fusion protein was purified by electric elution method, and its antigenicity was examined by Western blotting and lymphocyte proliferation test. Results The gene of miracidial antigen was cloned into the expression plasmid pGEX-4T-3. After induced by IPTG, the recombinant expressed a fusion protein of GST-SjMP10, with a molecular weight of 39 000 approximately. The purified fusion protein showed proper antigenicity that could be recognized by the sera of rabbits heavily infected by Schistosoma japonicum and could stimulate the proliferation of splenic lymphocytes of infected BALB/c mice. Conclusion The miracidial antigen from eggs of Schistosoma japonicum was expressed successfully.

Objective To analyze the full length cDNA sequence of Schistosoma japonicum egg miracidia genes harboring signal sequence.Methods The gene specific primers were designed and synthesized according to S.japonicum egg miracidia cDNA fragment containing signal sequence identified by signal sequence trapping method previously. The 5′and 3′ end cDNA fragments of each egg miracidia cDNA fragment harboring signal sequence were amplified by nest PCR using the first strand cDNA of S.japonicum as the template. The specific PCR fragments were cloned by TA clone method and sequenced. The full length cDNA sequence of each gene with signal sequence was constructed by comparing the cDNA sequence identified with signal sequence trapping method and the 5′ end sequence, the 3′ end sequence and deleting the overlapping fragments. The splicing model between mRNA of signal sequence and one of mature portions of S.japonicum egg miracidia gene was checked by analyzing the genomic DNA sequence structure of some genes with signal sequence. Results The 5′ and 3′ end cDNA fragments of sixteen among thirty cDNA fragment with signal sequence were amplified successfully, and their DNA sequences were determined. The full length cDNA sequences of sixteen egg miracidia genes were obtained by sequence matching and splicing. The results of deduced amino acid analysis found that the signal peptide of gene SjP4001 was the same to the one of SjP1531 and the signal peptide of gene SjP1183 was similar to the one of gene SjP3742. It confirmed that different genes could share the same or similar signal peptide. The data of S.japonicum genomic DNA sequence analysis showed that the S.japonicum could obtain its signal sequence by alternative splicing model or trans-splicing model. Conclusions The full length cDNA sequences of sixteen S.japonicum egg miracidia genes with signal sequence have been defined, it indicated that the S.japonicum egg miracidia genes could get its signal sequence by alternative splicing model or trans-splicing model was found in this study.

Objective To prepare a novel nano-suspension of niclosamide ethanolamine and evaluate its molluscicidal effect. Methods Niclosamide ethanolamine and stabilizing agent—polyvinylpyrrolidone (PVP) were dissolved in dimethyl sulfoxide. After the solution was added into water under fast stirring, niclosamide ethanolamine was quickly precipitated to form nanoparticles and nano-suspension was obtained. The weight ratio of PVP to niclosamide ethanolamine, concentration, temperature, stirring speed on the size and distribution of the nanoparticles were investigated. The molluscicidal effect of niclosamide ethanolamine nano-suspension was measured by immersion and spray methods in the laboratory and field. Results When the weight ratio of PVP to niclosamide ethanolamine was from 1∶2 to 1∶3, the nanoparticles of the niclosamide ethanolamine had diameters about 100 nm and the nano-suspension was stable without agglomerating for more than 1 month; as the speed of the stirring increased, the nanoparticles prepared became smaller and more stable. LC50 of the nano-suspension was 0.0544 mg/L but the LC50 of wettable powder of niclosamide ethanolamine salt (WPN) was 0.1250 mg/L. In the field immersion and spray tests, the concentration of nano-suspension as only 1/5 of active content of WPN achieved the same molluscicidal effect with WPN. Conclusions The nano-suspension has higher molluscicidal effect than WPN and the novel formulation of niclosamide has more advantages than WPN, it is useful for snail control in the field.

Objective To investigate the value of recombinant fusion protein (GST-HD)of the large hydrophilic domain (HD) of 23 kDa membrane protein of Schistosoma japonicum with the Glu-tathione-S-transferase (GST) of S. japonicum for early diagnosis of schistosorniasis. Methods The rabbits were infected with cercariae of S. japonicum Chinese mainland strain (300 per one). The rabbits' sera before infection and after being infected at different time were collected. The antibodies IgG(s) against recombinant GST-HD and SEA were measured respectively by ELISA to observe the rabbits' immune reaction status to GST-HD and SEA at different time after being infected with the cercariae. At the same time, 90 serum samples of patients with acute schistosorniasis and 30 samples of healthy persons were checked with GST-HD to evaluate its value for early diagnosis of schistosorniasis. Results At the 17th, 21st and 24th day after infection, the positive rates of antibody IgG of rabbits sera against GST-HD were 42.85%, 92.80% and 100.00% respectively, but the positive rates of antibody IgG against SEA were 14. 28% , 50. 00% and 84.60% respectively. The sensitivity of GST-HD for detecting early schistosome infection was higher than that of SEA significantly. The predictive values of positive and negative of GST-HD for detecting acute schistosorniasis was 98. 89% and 96.67%,respectively, and the diagnostic efficacy was 98. 33%. Conclusion The recombinant GST-HD fusion protein has high early diagnostic value for schistosorniasis.

Objective To construct and evaluate a novel PAMAM dendrimer vector-DNA vaccine for schistosomiasis japonica.Methods Lysine was used to modify 4.0G PAMAM.and the modified product PAMAM-Lys was synthesized.Agarose gel electrophoresis was used to confirm the composite ratio of plasmid DNA and dendrimer.Micrestructure of the compound was observed by using transmission electronic microscopy,and the stability was analyzed by using electrophoresis.The viability of the cells transfected with dendrimers was evaluated by using a MTT technique in vitro.Fiftyty mice were immunized with purified plasmid pJW4303,pJW4303-Sj23 dendrimer PAMAM-Lys and compound PAMAM-Lys/pJW4303-Sj23,respectively.The specific antibodies of the mice in each group were detected to access the immunoreactivity.Results The agarese gel electrophoresis showed that when the charge ratio of the dendrimer vector and DNA was between 2 and 4,the positive and negative charges could be counteraeted completely,and the compound was blocked completely by DNA electrophoresis.The obscrvation results with transmission electronic microscopy showed that the composition of dendrimer vector and DNA caused shrink of DNA structure.Dendrimer-DNA compound had a good stability.MTT showed the modified dendrimer vector and DNA compound system produced a lower cell toxicity on 293T cell than the unmodified Ones.Thk levels of specific antibodies of the mice immunized with PAMAM-Lys/pJW4303Sj23 were significantly higher than those of the mice immunized with naked DNA vaccine(P<0.05).Conclusions The lysinemodified PAMAM-lys is an excellent vector,and has an appropriate biocompatibility.Lysine-modification can reduce the cell toxicity of PAMAM dendrimer significantly.PAMAM-Lys can enhance the immunoreactivity of DNA vacmine which merits further application in schistosomiasis DNA vaccine.

This study reported the primary application of FA-ELISA from 107-121kDa fration antibody of Schistosoma Japonicum for detection of the short-term antibody in patients with schistosomiasis. The result showed that this method provided high sensitivity (with positive rate of 93. 33% with 30 cases of schistosomiasis) and good specificity (no false postive in 60 health objects). When one group of schistosome patients were examined with FA -ELISA and with the routine SEA-ELISA before chemotherapy and at 8 months,1 year and 1. 5 year after treatment,it showed good property for evaluation of chemotherapy efficacy with FA -ELISA,which was much better than the routine method.

Objective To evaluate the stability of dipstick dye immunoassay (DDIA) kit forschisitosomiasis diagnosis. Methods By means of detection of the sera from infected people withSchistosoma japonicum and healthy people, the stability of the DDIA kit, which stored at 37℃,room temperature or 4 ℃ respectively, was evaluated depending on the detective results ofsensitivity, specificity, detectable minimum and coefficient variation ( CV). Results Thesensitivity, specificity, detectable minimum and coefficient variation of the DDIA kit were invariableafter the kits stored at 37 ℃ for 180 days, and at room temperature or 4 ℃ for 360 days.Conclusion The DDIA kit is stable while it stores at 37℃ for 180 days, and at room temperatureor 4℃ for 360 days at least.