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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.
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<p><b>OBJECTIVE</b>To identify the egg antigens related to the formation of hepatic granulomas and fibrosis of Schistosomiasis japonica.</p><p><b>METHODS</b>The egg cDNA library of Schistosoma japonicum (S. japonicum) was constructed and screened by immunological methods with the pooled sera of advanced schistosomiasis patients. The inserted foreign DNA fragments of positive clones were sequenced. The sequence data were analyzed using Wdnasis 2.5 and compared with Genebank data using blast software.</p><p><b>RESULTS</b>Eighty-one clones containing recombinant DNA fragments were obtained from the egg cDNA library of S. japonicum by immunological screening. The DNA sequences of all clones belonged to the miracidial antigen family. The longest cDNA fragment was 1604 bp, which contained an open reading frame of 351 bp, which encoded a protein of 1 2913.35 daltons.</p><p><b>CONCLUSION</b>The cDNA sequence of the miracidial antigen of S. japonicum (Chinese strain) was obtained for the first time.</p>
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Animals , Antigens, Helminth , Genetics , Base Sequence , China , DNA, Complementary , Ovum , Schistosoma japonicum , Genetics , Allergy and ImmunologyABSTRACT
Objective To evaluate the toxicity of suspension concentrate of niclosamide(SCN)for molluscicide in the field.Methods According to the state standard of the People's Republic of China "The methods of toxicity test for agriculture register",GB15670-1995,the experiments of acute toxicity on rats and fish were carried out.Results LD50(s)of SCN via mouth and skin with rats were more than 5 000 mg/kg respectively,and LC50(s)of SCN via inbreathe with rats were more than 5 000 mg/m3.Based on the classification of appraising criterion on acute toxicity test,it belonged to a feebleness toxicity degree.The eye and the skin stimulating tests with rabbits showed that it did not irritate the eyes and the skin.For fish,its acute toxicity was slightly lower than that of pure niclosamide,and markedly lower than that of pure niclosamide ethanolamine salt and WPN.Conclusions SCN belongs to a feebleness toxicity degree and has a lower toxicity to fish.It should be a useful molluscicide in endemic areas of schistosomiasis.
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Objective To develop a novel synergism compound suspension concentrate of niclosamide and chlorphoxim (Co-SCN) and sdudy its characteristics. Methods Niclosamide and chlorphoxim were milled by a ball mill and mixed with different amounts of wetting agent. disper-sant agent, thickener, and water etc. , to develop Co-SCN, and the pH value, thickener, grain size were evaluated. The ultraviolet absorption spectrum of niclosamide and chlorphoxim were measured. The content of niclosamide and chlorphoxim in the solution were assayed by HPLC. Results Co-SCN was a gray thickener fluidity liquid. It was very easy to disperse and could be mixed with water in any proportion. Its pH was 8. 65 and thickener was 137 mpa? s. The grain sizes (diameter) were from 0. 138-19. 953 ?m. Of them more than 95. 6% was smaller than 10 ?m and more than 82. 24% was smaller than 5 ?m. There were three peaks of ultraviolet absorption spectrum for niclosamide: 210, 234 nm and 334 nm respectively. One peak of chlorphoxim was at 269 nm. The novel formulation contained 20.64% niclosamide and 5.26% chlorphoxim. The suspension stability of Co-SCN was 100% for 2 hours and 89. 14% for 4 hours, and otherwise WPN in water was speedy sediment. Conclusion The novel synergism compound suspension concentrate of niclosamide and chlorphoxim is a stable quality and standard formulation.
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Objective To evaluate the molluscicidal effect of colistin E on Oncomelania hupensis. Methods The molluscicidal effect of colistin E on O. hupensis was checked by using the immersion method and spraying method. The toxicity of colistin E on fish was observed by using the toxic test. Results The snail mortality of each group was 100% when the snails were immersed in the colistin E solutions at a concentration of 5. 0, 1. 0 g/L for 24, 48 h and 72 h separately. When the snails were immersed in the colistin E solution at a concentration of 0. 5 g/L for 24, 48 h and 72 h, the death rates were 95% , 100% and 100% respectively; when the snails were immersed in the colistin E solution at a concentration of 0. 1 g/L for 24, 48 h and 72 h, the mortality was 90%, 95% and 100% respectively; when the snails were immersed in the colistin E solution at a concentration of 0. 01 g/L for 24, 48 h and 72 h, the mortality was 70%, 86% and 100% respectively. The snail mortality by the spraying method in a dose of 35, 70, 140 mg/m2 of colistin E was 60%, 100% and 100%. The result of toxic test showed that the toxicity of colistin E on fish was low. Conclusion Colistin E is an effective molluscicide, and is worthy of investigation further.
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Objective To identify the trend of endemic situation among surveillance sites in Jiagsu Province from 2000 to 2002. Methods Twelve schistosomiasis surveillance sites were es-tablished ,and the longitudinal, surveillance was carried out. Results The related index of snail increased in most of surveillance sites, the rates of positive snails rose rapidly in marshlands. The infection rates of Schitosoma janponicum of cattle decreased and infection rates of human were relatively steady. However, there was still the danger of heavy endemic. Conclusion Current control strategies can not effectively adapt to the endemic situation of schistosomiasis, although which have some effects on control of morbidity. We need to study the new characteristics and rule of the endemic of schistosomiasis, and make out more effective control strategies which can suit with the current society, economies and nature environment.
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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.
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Objective To screen the bacteria and its components which are toxic to Oncomelania hupensis. Methods The samples of Oncomelania hupensis on the point of death and the soil around the snails were collected. The bacteria existing in the snails and soil were isolated and identified by using regular methods. After being fermented, the toxicity of the bacterium components including the ferment supernatant, split products and bacteria to the snail were tested by the toxicity test. Results Totally, 104 strains of bacteria were isolated from the snails and soil samples, which included Gram positive cocci or bacilli, Gram negative cocci or bacilli. The fermenting supernatant, splitting products and 10 strains of bacteria showed different level of toxicity against the snail respectively. All the fermenting supernatant of bacterium PY1-1, PY7-2, PY3-5, PY19-3, PY2-2-2, PY8-2-2, PY16-1 could kill more than 50 percent of snails. Conclusion The bacteria which are poisonous to snails have been isolated that make a good basis for identifying single toxic component against snails.
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Objective To construct, express and purify human scFv antibody (S1) against the recombinant SAG1 of Toxoplasma fused to magainin, and observe its protective effect against Toxoplasma in infected mice. Methods The S1 scFv antibody gene amplified from phagmid S1/pIT-2 fused to magainin was cloned into procaryotic expression vector pET-32c. The recombinant plasmid S1M/pET-32c proved by DNA sequencing was transformed into E.coli BL21, and induced for fusion expression of S1M with IPTG. The expressed S1M was purified with Ni 2+ chelating HiTrap HP column and detected with SDS-PAGE. The effect of reduction of infection of Toxoplasma was observed through in vivo and in vitro experiments in mice. Results The fused gene of S1 and magainin was successfully cloned into procaryotic expression vector pET-32c proved by DNA sequencing. The recombinant S1M protein about 43 kDa was expressed in E.coli as inclusion body, and prepared with Ni 2+ column purification. Tachyzoite of Toxoplasma preincubated with S1M showed decreased infectivity in mice, the result of in vivo experiments showed that mice treated with S1M hadlonger survival time than the mice untreated. Conclusion The purified targeting antimicrobial peptide S1M could reduce the infectivity of tachyzoites of Toxoplasma in a certain extent and has a potential value for biological therapy of toxoplasmosis; otherwise, the constructed targeting antimic robial peptide S1M also provides a new model for biological therapy of toxoplasmosis.
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Objective To design the Internet geographic information system (GIS) on schistosomiasis of Jiangsu Province using the technology of WebGIS. Methods Based on the GIS database of schistosomiasis, the active model of WebGIS on schistosomiasis was developed with the software of ArcIMS. Results The WebGIS of schistosomiasis has been developed and it has been running on the intranet of Jiangsu Institute of Parasitic Diseases. The system would manage the geographic database and attribute material database, and it would provide the function of query, display, analysis, statistics, export, etc. Conclusion It is feasible to design the WebGIS of schistosomiasis, which provides the basics of developing the Management Decision Systems of Schistosomiasis of Jiangsu Province.
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Objective To understand whether the breeding time of snails influences the evaluation on molluscicidal efficacy against Oncomelania snails so as to make a standardization of methods for screening molluscicides in laboratory. Methods Snails collected from the endemic areas in Nanjing and Tongling cities bred for 1,6,11,16,21.31,61 and 91 days respectively, and then were immersed in 1.000 0,0.500 0,0.250 0,0.125 0,0.062 5 and 0.031 3 mg/L solution of niclosamide for 24 and 48 hours at 25℃ in laboratory. Results One hundred percent killing rate was achieved in the groups of the snails bred for 1 - 11 days and immersed in 1.0 mg/L niclosamide for 24 hours. The LC_(50) concentrations of snails collected from Nanjing, Jiangsu Province and immersed for 24 hours varied from 0.094 7 to 0.133 9 mg/L, and for 48 hours from 0.071 8 to 0.092 6 mg/L.Those of snails collected from Tongling, Anhui Province for 24 hours varied from 0.082 5 to 0.103 9 mg/L, and for 48 hours from 0. 071 8 to 0.082 5 mg/L. The molluscicidal effect was unstable in the groups of snails bred for more than 16 days. There was a significant difference (X_(Nanjing24 h)~2 = 22.26,P
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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.
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Objective To enhance the protective immunity effects of nucleic acid vaccines against Schistosoma japonicum infection by electroporation(EP)in vivo in infected BALB/c mice.Methods Plasmids and proteins for immunization were prepared and diluted in no bacterial saline solution to final concentration of 1.5 mg/ml.pcDNA3.1-SjC23,pcDNA3.1-SjCTPI,pcDNA3.1-(CDR3)6 plasmid DNAs were mixed by equal volume to form the cocktail DNA vaccine,and also mixed with recombinant proteins SjC23-HD,SjCTPI,and NP30 by equal volume to form the cocktail protein vaccine.Seventy female BALB/c mice of 4-5 weeks old were randomly divided into 5 groups(A,B,C,D,E).In Group A(control group),each mouse was immunized with 100 ?l saline solution by intramuscular(i.m.);in Group B(pcDNA3.1/EP control group),each mouse was immunized(i.m.)with 100 ?l pcDNA3.1 followed by EP in vivo for three times at week 0,3,6;in Group C(pcDNA3.1/EP plus cocktail protein vaccine group),each mouse was immunized(i.m.)with 100 ?l pcDNA3.1 followed by EP for three times at week 0,3,6 and boosted with 100 ?l cocktail protein vaccine plus 100 ?l FCA by subcutaneous at week 9;in Group D(cocktail DNA vaccine/EP group),each mouse was immunized(i.m.)with 100 ?l cocktail DNA vaccine followed by EP for three times at week 0,3,6;in Group E(cocktail DNA vaccine/EP plus cocktail protein vaccine group),each mouse was immunized(i.m.)with 100 ?l cocktail DNA vaccine followed by EP for three times at week 0,3,6 and boosted with 100 ?l cocktail protein vaccine plus 100 ?l FCA by subcutaneous at week 9.Four weeks after the last DNA immunization or two weeks after protein boosting,all the mice were challenged with(40?1)cercariae of Schistosoma japonicum by abdominal skin penetration.Forty-two days post-challenge,the mice were sacrificed and perfused,and the numbers of recovered worms and eggs in liver were counted.The blood was collected from the tail veins of all the mice two days before the first immunization and challenge,respectively,the serum was prepared for detection of IgG,IgG1 and IgG2a.Two days before the challenge,the spleen cells of two mice from each group were cultured and stimulated with ConA and soluble egg antigen(SEA),and the supernatant was collected for detection of IL-2,IL-4 and IFN-? by flow cytometre.Results The worm reduction rates in Group C,D and E were 18.09%,45.00% and 57.09%,respectively,compared with the control group.The worm reduction rates in Group D and E were significantly higher than that in Group C(P
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Objective To enhance the protective immunity effects against Schistosoma japonicum infection by priming with cocktail DNA vaccines and boosting with cocktail protein vaccines in infected BALB/c mice.Methods Plasmids and proteins for immunization were prepared and diluted in no bacterial saline solution to final concentration of 1.5 mg/ml,and mixed with pcDNA3.1-SjC23,pcDNA3.1-SjCTPI,pcDNA3.1-(CDR3)6 plasmid DNAs by equal volume to form the cocktail DNA vaccine,and also mixed with recombinant proteins SjC23-HD,SjCTPI,and NP30 by equal volume to form the cocktail protein vaccine.Seventy female BALB/c mice of 4-5 weeks old were randomly divided into 5 groups(A,B,C,D,E).In Group A(control group),each mouse was immunized with 100 ?l saline solution by intramuscular(i.m.);in Group B(pcDNA3.1 control group),each mouse was immunized(i.m.)with 100 ?l pcDNA3.1 for three times at week 0,3,6;in Group C(pcDNA3.1 and cocktail protein group),each mouse was immunized(i.m.)with 100 ?l pcDNA3.1 for three times at week 0,3,6 and immunized with 100 ?l mixed protein vaccines plus 100 ?l FCA by subcutaneous at week 9;in Group D(cocktail DNA vaccines group),each mouse was immunized(i.m.)with 100 ?l mixed DNA vaccines for three times at week 0,3,6;in Group E(cocktail DNA vaccines plus cocktail proteins),each mouse was immunized(i.m.)with 100 ?l mixed DNA vaccines for three times at week 0,3,6 and immunized with 100 ?l mixed protein vaccines plus 100 ?l FCA by subcutaneous at week 9.Four weeks after the last DNA immunization or two weeks after protein boosting,all the mice were challenged with(40?1)cercariae of Schistosoma japonicum by abdominal skin penetration at the same time.Forty-two days post-challenge,the mice were sacrificed and perfused,and the numbers of recovered worms and eggs in liver were counted.The blood was collected from the tail veins of all the mice two days before the first immunization and challenge,respectively,the serum was prepared for detection of IgG,IgG1 and IgG2a.Two days before the challenge,the spleen cells of two mice from each group were cultured and stimulated with ConA and soluble egg antigen(SEA),and the supernatant was collected for detection of IL-2,IL-4 and IFN-?.Results The worm reduction rates in Group C,D and E were 17.70%,32.88% and 45.35%,respectively,compared with the control group.The worm reduction rates in Group D and E were significantly higher than that in Group C(P
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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.
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Objective To screen and identify B cell epitopes in SAG1, SAG2, SAG3, GRA1, GRA6 and P35 antigens of Toxoplasma gondii. Methods The indexes such as hydrophilicity, accessibility, flexibility, secondary structure and polarity of the 6 antigen moleculars above mentioned were analyzed by BioSun system. Two B cell epitopes with high antigenicity from each antigen molecular were selected, and the total twelve pairs of oligonucleotide chains were designed according to the 12 B cell epitopes’ sequence and synthesized, then cloned into plasmid pET-32c. The 12 fragment B cell epitopes were expressed and the expressed fusion proteins were identified with Western blot. Results Twelve B cell epitopes from 6 Toxoplasma antigens (two from each antigen) were predicted and selected. The epitope genes were successfully cloned into pET-32c and expressed. Western blot results showed that 3 of 12 expressed fusion proteins could be recognized by the immunized rabbit sera with soluble antigen of Toxoplasma gondii, but not by the unimmunized rabbit sera Conclusion Three B cell epitopes of Toxoplasma[with potential diagnostic value are obtained.
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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.
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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.
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Objective To predict B cell epitopes in Sj22, Sj23, Sj14-3-3, Sj26 of Schistosoma japonicum with bioinformatics, and evaluate the antigenicity of these epitope proteins. Methods The complete DNA sequences of S.japonicum were predicted by BioSun system, the target B cell epitope genes were selected, cloned and expressed. The expressed fusion proteins were detected with the sera of schistosomiasis patients and health people for evaluation of their antigenicity. Results Eight B cell epitopes from four molecules of S.japonicum were predicted. The B cell epitopes of Sj22 probably located in 56-62 and 127-133 amino acids. The B cell epitopes of Sj23 probably located in 149-156 and 160-167 amino acids. The B cell epitopes of S14-3-3 probably located in 118-125 and 130-137 amino acids. The B cell epitopes of Sj26 probably located in 143-149 and 191-197 amino acids. The predicted epitope genes were cloned into pET-32c plasmid and expressed. Three of eight expressed fusion proteins of epitopes were reacted with the sera of schistosomiasis patients but not with health people. Conclusion Three epitope antigens with potential diagnosis value are determined.
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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.