Genome-wide miRNAs expression profiles of Schistosoma japonicum schistosomula in response to artesunate.

AIM: miRNAs play a significant role in pharmacogenomics and are likely to be important in the molecular mechanism of atesunate (ART) effects on Schistosoma japonicum. METHODS: We sequenced the RNAs using an Illumina (Solexa) DNA sequencer and compared the relative expression levels of the miRNAs in 10-day-old schistosomula from ART and the parallel control group. RESULTS: We characterized 95 known miRNAs from S. japonicum schistosomula individuals, including 38 novel miRNA families. Among the detectable 134 miRNAs differentially expressed (>2.0-fold change, p < 0.01) after ART treatment in schistosomula, a total of seven known or novel 3p- or 5p- derived S. japonicum miRNAs were characterized. We propose that sja-miR-125b may regulate the expression of ART metabolizing enzymes, glutathione synthetase or heme-binding protein 2 to help S. japonicum resists or adapts to drug stress and also ART may significantly inhibit sexual maturation of female worms mediated by mir-71b/2 miRNA cluster. CONCLUSION: This was the first comprehensive miRNAs expression profile analysis of S. japonicum in response to ART, and provides an overview of the complex network of the mechanism of action of ART on S. japonicum.

Development of a rapid dipstick with latex immunochromatographic assay (DLIA) for diagnosis of schistosomiasis japonica.

BACKGROUND: Schistosomiasis japonica (schistosomiasis) is a zoonosis that can seriously affect human health. At present, the immunodiagnostic assays for schistosomiasis detection are time-consuming and require well-trained personnel and special instruments, which can limit their use in the field. Thus, there is a pressing need for a simple and rapid immunoassay to screen patients on a large scale. In this study, we developed a novel rapid dipstick with latex immunochromatographic assay (DLIA) to detect anti-Schisaosoma japonicum antibodies in human serum. RESULTS: Using latex microspheres as a color probe, DLIA was established to test standard positive and negative sera, in comparison with the classical enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity of DLIA were 95.10% (97/102) and 94.91% (261/275), respectively. The cross-reaction rates with clonorchiosis, intestinal nematodes, Angiostrongylus cantonensis and paragonimiasis were 0, 0, 0 and 42.11% respectively. All the results showed no significant difference to the ELISA. In field tests, 333 human serum samples from an endemic area were tested with DLIA, and compared with ELISA and Kato-Katz method. There was no significant difference between DLIA and ELISA on positive and negative rates of detection; however, significant differences existed between DLIA and Kato-Katz method, and between ELISA and Kato-Katz method. The kappa value between DLIA and ELISA was 0.90. CONCLUSIONS: This is the first study in which DLIA was used to detect anti-Schistosoma japonicum antibody. The results show that DLIA is a simple, rapid, convenient, sensitive and specific assay for the diagnosis of schistosomiasis and is therefore very suitable for large-scale field applications and clinical detection.

[A study of double antigen sandwich colloidal gold immunochromatography rapid detection for Mycobacterium tuberculosis antibody].

OBJECTIVE: To establish a simple, fast and accurate double antigen colloidal gold immunochromatographic technique for detecting Mycobacterium tuberculosis antibody from tuberculosis patients. METHODS: The fusion protein ESAT-6-16-38 was constructed by using gene cloning technique for the 6, 16 and 38 kDa early secreted antigenic target from Mycobacterium tuberculosis. The ESAT-6-16-38 fusion protein was marked to colloidal gold to establish the double antigen colloidal gold immunochromatographic assay. Serum samples from 163 patients with tuberculosis, including 57 sputum-positive cases, 64 sputum-negative cases, and 42 cases with extrapulmonary tuberculosis, were collected during 2007 and 2009 from the Disease Prevention and Control Center of Deqing County. In addition, 573 controls (224 healthy volunteers, 217 patients with acute pneumonia and bronchitis, 132 patients with paragonimiasis) were recruited for comparison. Mycobacterium tuberculosis specific antibodies were detected by using immunochromatographic and protein chip technique. Detection rate was compared with Chi-square test. RESULTS: Among the 163 tuberculosis patients, the positive rates of immunochromatographic detection and protein chip were 73.0% (120/163) and 72.4% (118/163) respectively; the difference was not statistically significant (χ()2 = 0.062, P > 0.05). Among the 573 controls, the negative rates of immunochromatographic detection and protein chip were 93.9% (538/573) and 92.0% (527/573) respectively; the difference was not statistically significant (χ()2 = 0.635, P > 0.05). There was no cross reaction in the paragonimiasis patients. The positive rate of the immunochromatographic assay was as high as 87.7% (50/57) in the sputum-positive patients. CONCLUSIONS: The double antigen immunochromatographic technique is an easy to operate, rapid, highly sensitive, specific, and reproducible method for the detection of Mycobacterium tuberculosis antibodies.

[Establishment and application of rapid dipstick of latex immunochromatographic assay for the diagnosis of schistosomiasis].

OBJECTIVE: To establish a simple and fast diagnostic assay for schistosomiasis. METHODS: Based on the immunochromatographic technique and the principle of indirect assay of ELISA, using soluble egg antigen (SEA) of Schistosoma japonicum and mouse anti-human monoclonal antibody labelled with red latex as color developing agents, a latex immunochromatographic assay (DLIA) was developed. Serum samples from 69 schistosomiasis patients were detected by DLIA. Tested were also 264 sera from healthy people, 15 sera from clonorchiasis patients, 8 sera from patients with angiostrongyliasis cantonensis, 11 sera from patients with intestinal nematode infection and 19 sera from paragonimiasis patients. ELISA was used as a parallel control. RESULTS: The sensitivity for detecting schistosomiasis antibodies with DLIA and ELISA was 94.2% (65/69) and 95.7% (66/69), respectively (chi2=0.15, P>0.05). The specificity in examining healthy persons was 97.4% (257/264) and 94.7% (250/264), respectively (chi2=2.43, P>0.05). No cross reaction was found with the sera of clonorchiasis, intestinal nematode infection and angiostrongyliasis. The cross reaction rate with paragonimiasis of the two assays was 42.1% (8/19) and 47.4% (9/19), respectively (chi2=0.11, P>0.05). CONCLUSION: DLIA is a simple, fast, sensitive and specific assay for the diagnosis of schistosomiasis.

[Observation on the change of anti-S. japonicum antibody level in population migrated from outside embankment to new town].

OBJECTIVE: To detect the change of the anti-S. japonicum antibody level after people migrated from outside embankment to newly established town. METHODS: Three pilot spots were established for the investigation: one spot that both inhabitancy and cultivation disuse (A), one spot that only inhabitancy disuse but farming continued (B) and the third one served as control (C). DIGFA and ELISA were used to detect the antibody level in the populations from 2002 to 2005. RESULTS: The positive rate of anti-S. japonicum antibody declined significantly from 6.63% to 3.52% by DIGFA and from 7.26% to 3.71% by ELISA at spot A (chi2=5.2625, P<0.05; chi2=6.3296, P<0.05, respectively). There was no significant difference on the positive rate of antibody in spots B and C. The average A450 value of ELISA in the three spots was statistically analyzed by One-Way ANOVA. It was only in spot B that the average A450 value declined from 0.182 in 2003 to 0.147 in 2005 (P<0.01). CONCLUSION: The anti-S. japonicum antibody level in human population has decreased at certain degree after they migrated from outside embankment to new town.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum.

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5 percent and 47.6 percent less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Evaluation on the applied value of the dot immunogold filtration assay (DIGFA) for rapid detection of anti-Schistosoma japonicum antibody.

The dot immunogold filtration assay (DIGFA) is a rapid technique for the detection of anti-Schistosoma japonicum antibody. Its sensitivity with regard to sera obtained from patients with acute or chronic schistosomiasis was shown to be 100 and 96.9%, respectively. The specificity when using sera of people living in an area non-endemic for schistosomiasis japonica was 100%. Cross-reaction rates for paragonimiasis and clonorchiasis patients were 14.3% and 0%, respectively. Parallel serum tests of 1091 residents from an area endemic for S. japonicum by means of DIGFA, enzyme-linked immunosorbent assay and indirect haemagglutination test resulted in positive rates of 9.3%, 11.5% and 11.0%, respectively. Thus, there was a high level of agreement between the sets of results (P>0.05). In conclusion, DIGFA holds considerable promise for rapid and accurate diagnosis of S. japonicum, as it does not require any specific instruments and can be applied with ease. DIGFA has therefore several advantages over conventional diagnostic approaches and is useful not only for screening and sero-epidemiological surveys in the field, but also in clinical settings.

[Protective immunity of the recombinant Schistosoma japonicum specific very low density lipoprotein binding protein as a vaccine candidate].

OBJECTIVE: To investigate the protective immunity against Schistosoma japonicum in mice immunized with recombinant specific very low density lipoprotein binding protein (SVLBP) and its potential as vaccine candidate. METHODS: Recombinant SVLBP antigen was over-expressed under IPTG induction and purified by Ni-NTA affinity chromatography. C57BL/6 mice were immunized three times with purified reSVLBP complexed with Freund's adjuvant, at biweekly intervals. Then 35+/-1 cercariae of S. japonicum were given to each mouse by abdominal skin 10 days after the 3rd immunization. 45 days later, all mice were sacrificed to collect adult worms and count liver eggs. serum samples were collected before immunization and after challenge respectively, and were probed the antigen-specific antibodies using a panel of ELISAs. RESULTS: The worm burden and the egg deposition in liver tissue were reduced by 33.4% and 47.6% respectively in the immunized group, in comparison with the adjuvant control group (P<0.05). Higher titer (>1:6 400) of total IgG was observed after challenge infection. The vaccinated mice developed significantly higher levels of IgG2a, IgG2b, IgG1 than those of control mice. CONCLUSION: The recombinant tegumental SVLBP antigen could induce partial protection against S. japonicum infection. These data demonstrate the potential of SVLBP as a schistosome vaccine candidate.