[Assessment of Serum IgG and Its Subclasses in Cystic Echinococcosis Patients and Its Application for Diagnosis].

OBJECTIVE: To assess the diagnostic performance of hydatid cyst fluid (HCF) in detecting the anti-HCF IgG and its subclasses IgG1, IgG2 and IgG4 in serum of cystic echinococcosis patients. METHODS: ELISA was used to measure IgG and its subclasses IgG1, IgG2, and IgG4 specific for Echinococcus granulosus HCF, in the sera of 37 cystic echinococcosis (CE) patients and 29 healthy subjects in Huan County, Gansu Province. The receiver operating characteristic (ROC) curves of the four antibodies were analyzed by MedCalc software, referenced with the gold-standard B ultrasonic imaging. The diagnostic performances between the four antibodies were compared using paired z statistics based on the areas under the curve (AUC), and the best diagnostic threshold was determined for each. The sensitivity and specificity for detecting the four types of antibodies were compared using Chi-square test. RESULTS: The AUCs for IgG and its subclasses IgG1, IgG2, and IgG4 were 0.722, 0.919, 0.712, and 0.835, respectively; the AUC of IgG1 was significantly higher than those of IgG (z = 3.629, P < 0.05) and IgG2 (z = 3.292, P<0.05). The sensitivity for detecting IgG, IgGl, IgG2, and IgG4 was 54.1%, 91.9%, 67.6%, and 75.7%, respectively; the sensitivity for IgGl was significantly higher than that for IgG (χ2 = 3.84, P < 0.05), IgG2 (χ2 = 6.80, P < 0.05), and IgG4 (χ2 = 10.16, P < 0.05). The specificity for the four antibodies was 89.7%, 82.8%, 72.4%, and 89.7%, respectively, and no significant difference was found between them. In addition, the sensitivity for detecting IgG4 antibody was significantly higher in CE I-III than in CE IV-V patients. CONCLUSION: The IgG1 antibody shows the highest detection sensitivity by HCF, thus having potential value in diagnosis of cystic echinococcosis.

[Cloning, expression and immunodiagnostic analysis of Schistosoma japonicum calcium-binding EF-hand domain containing protein].

OBJECTIVE: To clone and expression Schistosoma japonicum calcium-binding EF-hand domain containing protein (SjEFCAB), purify the expressed protein, and evaluate its antigenicity and diagnostic value. METHODS: The positive clone screened from egg cDNA library was used as template to amplify the SjEFCAB gene by PCR. The target fragment was cloned into prokaryotic expression vector pGEX-4T-1. The positive recombinant plasmids were transformed into E.coli BL21 and induced by IPTG for expression of the protein. The recombinant protein was purified with GST-tag affinity chromatography. Western blotting was used to analyze the antigenicity. The purified protein was used as coating antigen for indirect ELISA to evaluate its diagnostic effect. Serum samples from patients with schistosomiasis japonica (78 cases), clonorchiasis sinensis (5 cases), cysticercosis (10 cases), paragonimiasis westermani (6 cases), trichinosis (9 cases) and healthy persons (50 cases) were examined. RESULTS: The recombinant plasmid pGEX-4T-1-SjEFCAB was constructed and the SjEFCAB recombinant protein (Mr 8 200) was expressed in E. coli. The soluble fusion protein was purified with affinity chromatography. Western blotting analysis showed that the recombinant protein was recognized by sera of infected rabbits and pooled sera of schistosomiasis japonica patients. The sensitivity and specificity of ELISA for diagnosis of schistosomiasis japonica were 82.1% (64/78) and 95.0% (76/80), respectively. The cross reaction with sera of clonorchiasis sinensis, cysticercosis, and trichinosis patients were 1/5, 1/10, and 1/9, respectively. There was no cross reaction with sera of paragonimiasis westermani patients. CONCLUSION: The recombinant SjEFCAB antigen has potential diagnostic value for schistosomiasis japonica.

[Immunoscreening of Schistosoma japonicum egg cDNA library and identification of positive clones].

OBJECTIVE: To obtain promising antigens for diagnosis or therapeutic efficacy assessment of schistosomiasis japonica. METHODS: At an endemic area in Anqing of Anhui Provinces, Kato-Katz method (six slides from two consecutive stool samples) was used to examine schistosome eggs and serum samples of 10 egg-positive patients were collected. The EPG of the ten patients, aged 13 to 64, was among 4 to 172. The patients were then treated with praziquantel 60 mg kg for two days. Sera of the same patients were collected again at six months post treatment. The egg cDNA library was immunoscreening with the pre- and post-treatment serum samples, and the positive clones were classified according to different reactions. The inserted fragments of positive clones were sequenced and analyzed for their homology through BLAST program. The amino acid sequences of the proteins were deduced from the gene sequences, and their structures and functions were predicted by bioinformatics tools. RESULTS: For the first round, 75 positive clones were screened from the egg cDNA library. Among them, 21 clones showed stronger reaction with the pre-treatment sera, and 8 clones showed stronger reaction with the post-treatment sera. According to the intensity of the reaction, types and the length of inserted fragments, 14 positive clones were selected for further study. Most of the clones belonged to miracidial antigen family, one clone was significantly homologous to the calcium-binding EF-hand, a domain-containing protein (score = 143), one was highly homologous to the cell wall integrity and stress response component 1 precursor (score = 487), and another four were homologous to the hypothetical protein. CONCLUSION: Twenty nine positive clones screened from the egg cDNA library show different reactions with the sera of schistosome-infected patients, before and after praziquantel treatment.

[Application and progress of fluorescence in situ hybridization in schistosome biology].

Schistosomiasis is one of the most serious parasitic diseases. Schistosome genes research provides the basis for study of schistosomiasis diagnosis, vaccine and drug targets. Fluorescence in situ hybridization (FISH) in schistosome focuses on researches of location of functional genes on chromosomes, genome physical mapping and chromosome identification. This article reviews the application of FISH in schistosome biology and its potential development.

Multiplex cytokine and antibody profile in cystic echinococcosis patients during a three-year follow-up in reference to the cyst stages.

BACKGROUND: Cystic echinococcosis (CE) is a worldwide parasitic zoonosis caused by infection of the larval stage of tapeworm Echinococcus granulosus. In human CE, the parasites develop and form cysts in internal organs. The differentiated cysts can be classified into five types based on WHO-IWGE standard CE1-5 representing different developmental stages. Infection with E. granulosus triggers hosts' humoral and cellular response, displaying elevated serum antibodies and Th1 and Th2 cytokines, which are presumed to be in association with the disease outcome. Identification of immunological markers for evaluation of disease progression has been a growing concern. However, the distinctive profile of cytokines and antibodies associated with the cyst progression has not been ascertained. METHODS: To better understand the interaction between host immune response and disease outcome, the present study followed-up four CE patients over three years by yearly measuring serum level of 27 cytokines, total IgG and isotypes, and ultrasound scanning, beginning in year 1 for all patients with CE1 and CE2 cysts before treatment and continued in year 2 with CE4 and in year 3 with CE3-CE5 post-treatment. RESULTS: Nine cytokines including Th1-type IL-2, Th17-type IL-17A, and inflammatory cytokines IL-1ß, IL-1Rα and TNF-α, chemokines IL-8, MIP-1α, MIP-1ß, and growth factor G-CSF were significantly elevated in patients with cyst type CE1, compared to the normal controls, and then declined to a normal level at CE4 and CE5. Examining the antibody production, we found that serum specific IgG was significantly increased in patients with active and transitional cysts, specifically the total IgG at CE1/CE3/CE4-CE5, IgG4 at CE1 and IgG1 at CE1/CE3 cyst status, in comparison with the normal controls, but showed no significant changes between the cyst stages. CONCLUSIONS: Our findings provide new information on the profile of multiplex cytokines and serum antibodies associated with cyst stages in cystic echinococcosis patients through a three-year follow-up, implying that further studies using an approach combining cyst-associated immune parameters may aid in identifying immunological markers for differentiation of disease progression.

[New polymorphic microsatellite loci identified using genomic resource for Schistosoma japonicum].

OBJECTIVE: To identify new microsatellite loci from genome sequence database for the study of polymorphism of Schistosoma japonicum. METHODS: Schistosoma japonicum isolates were obtained from seven endemic sites in China: Tongling and Guichi counties of Anhui Province, Duchang county of Jiangxi Province, Changde and Yueyang Cities of Hunan Province, Shashi City of Hubei Province, Xichang City of Sichuan Province. In order to study the genetic variance, genomic DNAs of 96 individual adult worms were screened against 17 new Schistosoma japonicum microsatellites and the raw data were analyzed by GenMapper 4.0. Furthermore, the varieties of alleles were inverstigated using GenA1Ex 6 and genetic distances within a subpopulation (GenClone) and among populations(UPGMA, MEGA 3.1) were analyzed. RESULTS: High levels of polymorphism were found between and within population samples, and significant genetic diversity was observed among the seven subpopulations. Within Jiangxi population, most genetic distances (17 loci) among samples range from 25 to 32, indicating a significant genetic diversity. There are three clusters among the seven populations: Jiangxi, Tonglin, Shashi and Changde population, with the genetics distances ranging from 0.0178 to 0.0363; Guichi and Yueyang population belong to another cluster, with the genetic distance of 0.0247; However, Xichang population is an unique group. Its genetic distances to other populations are notable with a range from 0.019 2 to 0.069 3. CONCLUSION: The 17 new polymorphic microsatellites identified may be used as suitable markers for the study on population genetics of Schistosoma japonicum and the genetic variance of the worms seems to be complicated.

Enzyme activity of Schistosoma japonicum cercarial elastase SjCE-2b ascertained by in vitro refolded recombinant protein.

Cercarial elastase (CE) secreted from cercariae is evinced to play a pivotal role in initial skin penetration of mammalian host. SjCE-2b, a Schistosoma japonicum CE orthologous to SmCE-2b in S. mansoni, was previously found present in cercarial stage to aid skin invasion, but its enzyme activity has not been validated due to the insolubility and altered conformation when expressed recombinantly in bacteria as inclusion bodies. We report here for the first time a bioactive and soluble recombinant SjCE-2b recovered successfully from inclusion bodies by refolding approaches, enabling our biochemical and immunological investigation of this enzyme. Using a "two-step-denaturing and refolding" method, we recovered an 83% yield with 90% purity of refolded protein. Proteolytic activity of rSjCE-2b was demonstrated and characterized by enzymatic assay, showing a Km of 0.116 mM and a specific activity of 1900 nmol p-nitroaniline/min/mg protein. A significant immunoprotective response was evidenced in mice immunized with refolded rSjCE-2b. The result of immunoprotection test is at apparent variance with previously reported findings using S. mansoni CE preparation, which was poorly immunogenic in immunized animals. This work extends the knowledge of schistosome cercarial protease, and presents a bioactive form of S. japonicum recombinant CE with high yield and good quality. This will allow further biochemical and biological investigations to explore schistosome CE activity and better understand the molecular mechanisms associated with cercarial skin invasion of the mammalian host.

An immunomics approach for the analysis of natural antibody responses to Plasmodium vivax infection.

High throughput immunomics is a powerful platform to discover potential targets of host immunity and develop diagnostic tests for infectious diseases. We screened the sera of Plasmodium vivax-exposed individuals to profile the antibody response to blood-stage antigens of P. vivax using a P. vivax protein microarray. A total of 1936 genes encoding the P. vivax proteins were expressed, printed and screened with sera from P. vivax-exposed individuals and normal subjects. Total of 151 (7.8% of the 1936 targets) highly immunoreactive antigens were identified, including five well-characterized antigens of P. vivax (ETRAMP11.2, Pv34, SUB1, RAP2 and MSP4). Among the highly immunoreactive antigens, 5 antigens were predicted as adhesins by MAAP, and 11 antigens were predicted as merozoite invasion-related proteins based on homology with P. falciparum proteins. There are 40 proteins that have serodiagnostic potential for antibody surveillance. These novel Plasmodium antigens identified provide the clues for understanding host immune response to P. vivax infection and the development of antibody surveillance tools.

An integrated immunoproteomics and bioinformatics approach for the analysis of Schistosoma japonicum tegument proteins.

Schistosomiasis remains one of the major neglected tropical diseases (NTDs) causing morbidity of humans residing in the tropical countries. Much effort has been devoted to the development of vaccines, since it is recognized that vaccines can be served as an important supplementary component alongside chemotherapy for the future control and elimination of schistosomiasis. To accelerate digging new potential target antigens, it is essential to extensively and intensively search immunogenic proteins in a high-throughput manner using proteomics-microarray techniques. In the present study, an integrated immunoproteomics and bioinformatics approach was used to profile the tegument of the human blood fluke Schistosoma japonicum. Results showed that the full-length tegument proteins were high-throughput cloned and expressed and screened with sera from S. japonicum-infected patients and normal subjects using protein arrays. Here, thirty highly immunoreactive tegument proteins and 10 antigens with an AUC value greater than 0.90 were identified at first time. In particularly, STIP1, the highest immunoreactive tegument protein has been shown good antigenicity and immunogenicity, and thus makes it to be a potential target for designing anti-parasite drug or vaccine. BIOLOGICAL SIGNIFICANCE: The schistosome tegument plays a crucial role in host-parasite interactions and there are several tegument proteins that proved to be potential vaccine candidates. However, vaccines are not yet available, thus it is important to identify new target antigens from schistosome tegument proteome. Herein, we demonstrate that the S. japonicum tegument proteins were analyzed by an integrated immunoproteomics and bioinformatics approach. We found that thirty highly immunoreactive tegument proteins and 10 antigens with an AUC value greater than 0.90 were identified for the first time. In particularly, we found 17 of tegument immunoproteomes having putative interaction networks with other proteins of S. japonicum. The results will provide clues of potential target molecules for vaccine development and biomarkers for diagnostics of schistosomiasis.

[Cloning, expression and bioinformatics analysis of cathepsin B of Echinococcus granulosus].

OBJECTIVE: To clone and express cathepsin B gene of Echinococcus granulosus (EgCatB) and analyze EgCatB protein by using bioinformatics tools and online databases. METHODS: The total RNA of E. granulosus was extracted and reversely transcribed into cDNA as the template sequence for PCR. The EgCatB gene was cloned by using the In-Fusion PCR cloning method and expressed by a wheat germ cell-free system, and then the recombinant protein was identified by Western blotting. The signal peptide, transmembrane helices and subcellular location of the EgCatB sequence were predicted by the online software SignalP 4.1, TMHMM sever v. 2.0 and TargetP 1.1 respectively. Subsequently, the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc software. Finally, the structures and the glycosylation modification site of the EgCatB encoding protein were analyzed and predicted in turn by ProtParam, SMART, Predictprotein, Swiss-model, NetOGlyc 4.0 and NetNGlyc 1.0 approaches. RESULTS: The EgCatB gene was successfully amplified from cDNA of E. granulosus and expressed in the soluble fractions. The molecular weight of the expressed protein was estimated 35 kDa. The bioinformatics analysis revealed that EgCatB was a classical secreted protein containing a Pept_C1 domain. The homology analysis indicated that the amino acid sequence of EgCatB was highly conserved in the active enzyme sites. The protein structure prediction showed a catalytic active center was formed through Gln106, Cys112, His282 and Asn302. It was found that there were nine O-glycosylation sites in the EgCatB sequence, but no N-glycosylation sites. CONCLUSIONS: The EgCatB gene is cloned and expressed successfully, and the recombinant protein is analyzed by bioinformatics approaches and structure predication. The study provides useful information for further functional study of the EgCatB protein.