Suppression of hyaluronidase reduces invasion and establishment of Haemonchus contortus larvae in sheep.

Haemonchus contortus is a hematophagous endoparasite of small ruminants, which is responsible for huge economic losses in livestock sector. Hyaluronidase produced by infective larvae of H. contortus can degrade hyaluronic acid present in the host's abomasal tissue. Thus, it facilitates larval tissue invasion and early establishment. We herein explored this ability of hyaluronidase in H. contortus, and tested whether hyaluronidase is utilized as a virulence factor by H. contortus while establishing the infection. We first successfully blocked the hyaluronidase gene in L3 larvae by RNA interference (RNAi), which was subsequently confirmed by qPCR, enzymatic activity, and immunohistochemistry assays. Using these larvae we then conducted in vivo and in vitro assays on sheep to assess the effects of hyaluronidase suppression on larval invasion and establishment of infection. The in vivo assay showed a significant drop in worm burden in siRNA treated group in comparison to control group. During in vitro assay we applied an ovine ex vivo model where siRNA treated group of larvae showed significantly reduced invasion of the abomasal tissue explants as compared to control group. These findings indicate that hyaluronidase plays a key role in host's tissue invasion and larval establishment, and it is used as a virulence factor by H. contortus while establishing the infection. As an invasive virulence molecule, its functional research is thus conducive to the prevention of haemonchosis.

Characterization and expression analysis of Wnt5 in Schistosoma japonicum at different developmental stages.

Wnt signaling is a key pathway involving the regulation of cell development and growth in metazoa. An analysis of Wnt signaling in Schistosoma japonicum might provide information regarding the molecular mechanisms underlying parasite development, which might be useful for vaccine screening and identification of pharmaceutical targets. The SjWnt5 gene, a member of the Wnt gene family, contained an 1149-bp open reading frame that encoded a 382-aa protein. Analysis of the SjWnt5 amino acid sequence revealed a domain that was conserved among members of the Wnt protein family. Expression of SjWnt5 was observed at all of the developmental stages in definitive hosts, and the highest level of SjWnt5 messenger RNA (mRNA) was detected at the schistosomula stage. Higher levels of SjWnt5 mRNA and protein were observed in mature male worms, compared with those in mature females. SjWnt5 mRNA was expressed at higher levels in maldeveloped worms from nonpermissive host or single-sex infection than in normal worms from permissive host and mixed-sex infection. The immunohistochemical analysis showed that SjWnt5 protein was expressed in the subtegumental musculature and acetabulum musculature of schistosomulum and adult worms, suggesting that SjWnt5 may play a role in regulation of parasite muscle development. Furthermore, SjWnt5 was found prominently expressed in the testes of the male and the ovary as well as the vitellarium of the female, suggesting that SjWnt5 may involve in the development of the reproductive organs of both sexes.

Evaluation of protective immune response in mice by vaccination the recombinant adenovirus for expressing Schistosoma japonicum inhibitor apoptosis protein.

Schistosomiasis is a worldwide parasitic disease, and while it can be successfully treated with chemotherapy, this does not prevent reinfection with the parasite. Adenovirus vectors have been widely used for vaccine delivery, and a vaccination approach has the potential to prevent infection with Schistosoma. Here, we developed a recombinant adenoviral vector that expresses Schistosoma japonicum inhibitor apoptosis protein (Ad-SjIAP) and assessed its immunoprotective functions against schistosomiasis in mice. Murine immune responses following vaccination were investigated using enzyme-linked immunosorbent assays (ELISA), lymphocyte proliferation, and cytokine assays. The protective immunity in mice was evaluated by challenging with S. japonicum cercariae. Our results indicated that immunization with the Ad-SjIAP in mice induced a strong serum IgG response against IAP including IgG1, IgG2a, and IgG2b. In addition, lymphocyte proliferation experiments showed that mice treated with Ad-SjIAP significantly increased the lymphocyte response upon stimulation with recombinant Schistosoma japonicum inhibitor apoptosis protein (rSjIAP). Moreover, cytokine assays indicated that vaccination of Ad-SjIAP significantly increased the production of interferon (IFN)-γ and IL-2 as compared to the corresponding control group. Furthermore, following the challenge with S. japonicum cercariae, the vaccine conferred moderate protection, with an average rate of 37.95% for worm reduction and 31.7% for egg reduction. Taken together, our preliminarily results suggested that schistosoma IAP may be a potential vaccine against S. japonicum and that adenoviral vectors may serve as an alternative delivery vehicle for schistosome vaccine development.

Effect of Ivermectin on the Expression of P-Glycoprotein in Third-Stage Larvae of Haemonchus contortus Isolated from China.

Haemonchus contortus poses a severe hazard to the healthy development of the sheep industry and threatens the welfare of sheep. Ivermectin is the primary anthelmintic used for the prevention and treatment of H. contortus parasitism. However, the widespread and uncontrolled application of ivermectin has resulted in the development and spread of resistant strains of H. contortus. P-glycoprotein (P-gp) plays important roles in the pharmacology and toxicology of ivermectin, and changes in P-gp expression levels can be used to analyze the resistance of H. contortus to ivermectin. This study aimed to analyze the effects of ivermectin on P-gp expression in H. contortus L3 larvae isolated from China and to evaluate whether changes in P-gp expression levels can be used to analyze resistant H. contortus strains. In the absence of drug treatment, the ivermectin-resistant strains isolated in China showed increased expression of P-gp11 (p < 0.01) compared with sensitive strains from elsewhere, whereas the expressions of P-gp2 and P-gp9.1 were downregulated (p < 0.01). When the same strain was compared before and after drug treatment, obvious differences in expression were observed between the different strains. Ivermectin-induced P-gp expression was found to be very complex among the L3 larvae of different strains. In addition, it was confirmed that using P-gp to determine ivermectin resistance in H. contortus strains from different geographic environments can yield different results.

Comparison of worm development and host immune responses in natural hosts of Schistosoma japonicum, yellow cattle and water buffalo.

BACKGROUND: Yellow cattle and water buffalo are two of the most important natural hosts for Schistosoma japonicum in China. Previous observation has revealed that yellow cattle are more suited to the development of S. japonicum than water buffalo. Understanding more about the molecular mechanisms involved in worm development, as well as the pathological and immunological differences between yellow cattle and water buffalo post infection with S japonicum will provide useful information for the vaccine design and its delivery procedure. RESULTS: The worm length (p < 0.01), worm recovery rate (p < 0.01) and the percentage of paired worms (p < 0.01) were significantly greater in yellow cattle than those in water buffalo. There were many white egg granulomas in the livers of yellow cattle, but fewer were observed in water buffalo at 7 weeks post infection. The livers of infected yellow cattle contained significantly increased accumulation of inflammatory cells, and the schistosome eggs were surrounded with large amounts of eosinophil infiltration. In contrast, no hepatocyte swelling or lymphocyte infiltration, and fewer white blood cells, was observed in water buffalo. The percentage of CD4⁺ T cells was higher in yellow cattle, while the percentage of CD8⁺ T cells was higher in water buffalo from pre-infection to 7 w post infection. The CD4/CD8 ratios were decreased in both species after challenge with schistosomes. Comparing with water buffalo, the IFN-γ level was higher and decreased significantly, while the IL-4 level was lower and increased gradually in yellow cattle from pre-infection to 7 w post infection. CONCLUSIONS: In this study, we confirmed that yellow cattle were more suited to the development of S. japonicum than water buffalo, and more serious pathological damage was observed in infected yellow cattle. Immunological analysis suggested that CD4⁺ T cells might be an integral component of the immune response and might associate with worm development in yellow cattle. A shift from Th1 to Th2 type polarized immunity was only shown clearly in schistosome-infected yellow cattle, but no shift in water buffalo. The results provide valuable information for increased understanding of host-schistosome interactions, and for control of schistosomiasis.

Analyses of mitogenomic markers shed light on the divergence, population dynamics, and demographic history of Pakistani chickens.

Pakistan is one of a few sites, associated with the earliest known independent domestication event in the evolutionary history of chicken, which is socio-economically and historically the most important poultry bird in the country. However, the divergence, past population dynamics, and demographic history of Pakistani chickens have not been addressed so far. Therefore, we herein investigated the indigenous Pakistani chickens using mitogenomic markers. We first prepared individual DNA samples from the chicken feathers, and generated nucleotide sequence data, which was then subjected to various population genetics analyses. In molecular phylogenetic analysis, the Pakistani chickens were clustered under nine different clades. Among the wild fowls, the Indian red jungle fowl (IRJF) shared very close affinities to Pakistani chickens. The Bayesian skyline plot showed an increase in the effective population size of Pakistani chickens during the last 50 years. Finally, a time-calibrated phylogeny inferred molecular divergence of the Pakistani chickens. A molecular rate of 3.6 × 10-6 mutations/site/year (95% HPD interval: 2.28 × 10-8 to 9.32 × 10-6) was estimated for the data set. In a rooted tree with root-age of 12058 years (95% HPD interval: 1161-38411), the Pakistani chicken haplotypes showed divergence from IRJF haplotypes around 6987 years (95% HPD interval: 1132-20746) ago, and they shared their most recent common ancestor with Gallus gallus spadiceus, and G. g. jabouillei at the root of the tree. Overall, these results suggest that Pakistani chicken haplotypes share their ancestral gene pool with the IRJF as compared to other red jungle fowl subspecies.

Molecular characterizations of an inhibitor of apoptosis from Schistosoma japonicum.

Apoptosis is a normal process for regulating cellular death of many organisms. Here, we molecularly characterized an inhibitor of apoptosis from Schistosoma japonicum (SjIAP). The transcription of the SjIAP predominantly occurred at the developmental stages in a final host. Functional assay indicated that the SjIAP could inhibit caspase activity either in 293T cell or in schistosome lysates. Additionally, there were differently expressed profiles of the SjIAP in S. japonicum living in different hosts. Our preliminary results suggest that the SjIAP may play important roles in parasitic living and development as well as in the host-parasite interactions, and drug target of SjIAP might be a potential for controlling schistosomiasis.

Molecular cloning and functional characterization of Schistosoma japonicum enolase which is highly expressed at the schistosomulum stage.

Enolase is a key enzyme in the glycolytic pathway; recent studies have also shown that enolase is found on the surface of several parasites, where it acts as a plasminogen-binding protein. In the present study, the enolase of Schistosoma japonicum has been cloned and expressed. In western blot analysis, the recombinant enolase from S. japonicum ( rSjENO) was recognized by rabbit sera directed against an antigen preparation from adult worms. Kinetic measurement revealed that rSjENO possesses good enzymatic activity. The real-time PCR showed that the enolase gene was highly expressed at 18-28 days of the life cycle. Immunofluorescence testing showed that SjENO was located mainly on the surface as well as in the inner tissues of the worms. Ligand-blotting analysis indicated that rSjENO could bind to human plasminogen as its receptor. In addition, a 24.28% reduction in the liver egg count and a reduction of 21.45% in the fecal egg count were observed in BALB/c mice vaccinated with rSjENO when compared with blank control mice. An ELISA assay suggested that high levels of specific IgG antibody could be induced by rSjENO in vaccinated mice.

Wnt4, the first member of the Wnt family identified in Schistosoma japonicum, regulates worm development by the canonical pathway.

The Wnt signaling pathway is an evolutionarily conserved signal transduction pathway used extensively during animal development. We aim, by increasing our understanding of the Wnt signaling pathway, to find a key gene or protein present in schistosomes that can be developed into vaccine candidate or drug target. We therefore isolated the Wnt4 gene from Schistosoma japonicum. Wnt4 encodes a putative protein of 558 amino acids which contains the conserved functional domain of the Wnt gene family. We suppressed the expression of Wnt4 mRNA in 10-day schistosomulae by RNA interference. Quantitative PCR analysis showed that Wnt4 displayed a 73% reduction in the transcript level. And GSK-3beta and beta-catenin, which are involved in Wnt canonical pathway, showed a 45% and 39% reduction in mRNA levels, respectively. PLC, CaMKII, DVL, and JNK, which are involved in Wnt non-canonical pathway, showed no reduction. These results suggest that the Wnt4 signal protein in S. japonicum regulates downstream genes by a canonical pathway. Wnt4 is the first member of the Wnt family to be identified in S. japonicum. An increased understanding of the Wnt signal transduction pathway will allow us to elucidate further the molecular mechanism of development in schistosomes.

A Whole Genome Re-Sequencing Based GWA Analysis Reveals Candidate Genes Associated with Ivermectin Resistance in Haemonchus contortus.

The most important and broad-spectrum drug used to control the parasitic worms to date is ivermectin (IVM). Resistance against IVM has emerged in parasites, and preserving its efficacy is now becoming a serious issue. The parasitic nematode Haemonchus contortus (Rudolphi, 1803) is economically an important parasite of small ruminants across the globe, which has a successful track record in IVM resistance. There are growing evidences regarding the multigenic nature of IVM resistance, and although some genes have been proposed as candidates of IVM resistance using lower magnification of genome, the genetic basis of IVM resistance still remains poorly resolved. Using the full magnification of genome, we herein applied a population genomics approach to characterize genome-wide signatures of selection among pooled worms from two susceptible and six ivermectin-resistant isolates of H. contortus, and revealed candidate genes under selection in relation to IVM resistance. These candidates also included a previously known IVM-resistance-associated candidate gene HCON_00148840, glc-3. Finally, an RNA-interference-based functional validation assay revealed the HCON_00143950 as IVM-tolerance-associated gene in H. contortus. The possible role of this gene in IVM resistance could be detoxification of xenobiotic in phase I of xenobiotic metabolism. The results of this study further enhance our understanding on the IVM resistance and continue to provide further evidence in favor of multigenic nature of IVM resistance.

[Immune protection of tegument protein rSj29 against Schistosoma japonicum in mice].

OBJECTIVE: To clone, express and characterize a tegument protein gene of Schistosoma japonicum (Sj29) , and investigate the immune protection of the recombinant protein against S. japonicum in mice. METHODS: The gene coding for Sj29 protein was amplified by PCR, and the sequence was analyzed by bioinformatics tools. Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced the recombinant with IPTG. The recombinant protein (rSj29) was purified by His-binding-resin affinity chromatography and characterized by Western blotting. Three groups each with 10 BALB/c mice were immunized subcutaneously three times (two weeks interval) respectively with 100 microl recombinant rSj29 (0.1 mg/ml) , adjuvant or PBS. At the 15th day after the final inoculation, each mouse was challenged by 40 +/- 2 cercariae of S. japonicum. At the 53rd day after infection, the mice were sacrificed to obtain the number of adult worms, number of eggs in liver and feces. Serum samples were collected at pre-immunization and certain time after immunization, and were analyzed for IgG by ELISA. The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique. mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR. RESULTS: A 576 bp Sj29 gene fragment was obtained. The recombinant protein rSj29 with Mr 22,900 was expressed in the form of inclusion body. The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice. The number of adult worms (15.4 +/- 5.9), number of hepatic eggs (40,143.3 +/- 2,995.9) and number of fecal eggs (3,803.9 +/- 110.9) in recombinant protein group were significantly higher than those of PBS control group (20 +/- 3.4, 49,318.1 +/- 6,648.3, 5,238.1 +/- 303.5, respectively) (P < 0.05) . There was a high level of specific IgG against rSj29 (maximum dilution 1:32000) in recombinant protein group. Immunohistochemical analysis showed the Sj29 protein expressed on the surface of different stages of S. japonicum. mRNA level of Sj29 was the highest at the 32nd day post-infection. CONCLUSION: The recombinant protein rSj29 induces certain degree of protective immunity in mice.

Ethnogenetic analysis reveals that Kohistanis of Pakistan were genetically linked to west Eurasians by a probable ancestral genepool from Eurasian steppe in the bronze age.

Despite the unique geographic, ethnic, social and cultural features of Kohistan in Pakistan, the origin and descent of Kohistanis remain still obscure. In an effort to address questions concerning the genetic structure, origin and genetic affinities of Kohistanis, we herein applied an ethnogenetic approach consisting on mitochondrial DNA (mtDNA) analysis and dental morphology analysis. We sequenced HVS1 of mtDNA, observed 14 haplotypes and assigned a total of 9 haplogroups belonging to macrolineages M (17%) and N (83%). Genetic diversity estimates in Kohistanis (Hd = 0.910 ±â€¯0.014; Pi = 0.019 ±â€¯0.001; θw = 0.019 ±â€¯0.006) were similar to that of previous studies in other Pakistani populations. Overall, the analyses of dental morphology and mtDNA profile of Kohistanis resulted in similar findings. All the analyses indicate that Kohistanis share affinities to populations from Europe, Near East, Central Asia and South Asia. The Kohistani HVS1 haplotype 2 shares 100% identity to HVS1 haplotypes across the Europe. These results in light of recent insights into ancient genomics lead us to conclude that ancestry from Eurasian Steppe genetically linked Kohistanis to all these populations in the Bronze Age. This is consistent with linguistic evidence and also with the Indo-Aryan migration model for the peopling of South Asia.

Population structure, genetic diversity and phylogenetic analysis of different rural and commercial chickens of Pakistan using complete sequence of mtDNA D-loop.

Chicken is the most important poultry bird in Pakistan that not only provides nutrition but also contributes to country's economy. The Pakistani chicken and its germplasm resources are not genetically characterized and explored. Here, we focused at rural and commercial chickens of Pakistan and explored genetic diversity, population structure and phylogeny. We first collected feather samples from Rural and Broiler populations of Pakistani chickens, isolated DNA and sequenced complete D-loop of mtDNA. The length of complete D-loop ranged from 1231 to 1234 bp in Pakistani chickens. The GC content was 39%. Hotspots of mutations were three hypervariable sites (HVS). Most of the variations (77%) were in HVS1. In a total, 26 polymorphic sites defined 12 haplotypes and all major haplogroups (A-I) in genetic structure of Pakistani chickens. Genetic diversity remained relatively very low in Broiler (Pi = 0.00212 ± 0.00136). There was a low sharing of matrilineages between the two populations (Fst = 0.170). With high Hd value (0.825 ± 0.051) and presence of all nine major haplogroups the rural chicken population showed relatively rich genepool. Finally we did molecular phylogenetic analysis and inferred phylogeny. Presence of subcontinent specific haplogroups E3 and I and clustering of Indian red junglefowl closely with Pakistani chickens in Bayesian inference tree, provide further evidence for an independent domestication event of chicken in subcontinent.

Microsatellite analysis reveals extensive gene flow, and lack of population structure in the farm populations of Haemonchus contortus in northern China.

The parasitic nematode Haemonchus contortus is economically an important parasite of small ruminants across the globe. China is the world's largest producer, consumer, and importer of mutton. With ubiquitous distribution across the country H. contortus is one of the potential candidates to cause huge economic losses to small ruminant farming industry in China. We herein investigated genetic diversity and population structure of six farm populations of H. contortus in northern China, and also compared them to H. contortus isolates from UK and Australia. We first prepared individual DNA samples from 240 adult worms, and generated genotyping data using eight microsatellite markers. Obtained data was then subjected to allelic frequency and population genetic analyses. The overall allelic richness (mean/locus/pop = 7.375 ±â€¯0.844-10.125 ±â€¯1.109), and expected heterozygosity (mean/locus/pop = 0.646 ±â€¯0.040-0.735 ±â€¯0.025) indicated high degree of population genetic variation across the Chinese isolates. Low level of genetic differentiation (Fst = 0.010-0.066) was observed across all the populations. AMOVA results showed high level of variation (93%) within the populations. PCA analysis revealed mixed clustering of all the populations with no visible geographical sub-structuring. Finally the population admixture analysis resulted in extensive admixing of genotypes across all the populations. With these findings we conclude that there is no obvious population genetic structure with extensive gene flow across all the farm populations of H. contortus in northern China.

Analysis of genome-wide SNPs based on 2b-RAD sequencing of pooled samples reveals signature of selection in different populations of Haemonchus contortus.

The parasitic nematode Haemonchus contortus is one of the world's most important parasites of small ruminants that causes significant economic losses to the livestock sector. The population structure and selection in its various strains are poorly understood. No study so far compared its different populations using genome-wide data. Here, we focused on different geographic populations of H. contours from China (Tibet, TB; Hubei, HB; Inner Mongolia, IM; Sichuan, SC), UK and Australia (AS), using genome-wide population-genomic approaches, to explore genetic diversity, population structure and selection. We first performed next-generation high-throughput 2b RAD pool sequencing using Illumina technology, and identified single-nucleotide polymorphisms (SNPs) in all the strains. We identified 75,187 SNPs for TB, 82,271 for HB, 82,420 for IM, 79,803 for SC, 83,504 for AS and 78,747 for UK strain. The SNPs revealed low-nucleotide diversity (pi= 0.0092-0.0133) within each strain, and a significant differentiation level (average Fst = 0.34264) among them. Chinese populations TB and SC, along with the UK strain, were more divergent populations. Chinese populations IM and HB showed affinities to the Australian strain. We then analysed signature of selection and detected 44 (UK) and 03 (AS) private selective sweeps containing 49 and 05 genes, respectively. Finally, we performed the functional annotation of selective sweeps and proposed biological significance to signature of selection. Our data suggest that 2b-RAD pool sequencing can be used to assess the signature of selection in H. contortus.

Spatiotemporal expression pattern of Sjfz7 and its expression comparison with other frizzled family genes in developmental stages of Schistosoma japonicum.

Wnts are secreted signaling molecules that are implicated in a variety of growth-related processes. Frizzled proteins have been identified as receptors for Wnt ligands in vertebrates and invertebrates, but a functional role for dioecious flatworm Frizzleds has not been determined. To evaluate the endogenous role of Frizzled proteins during development, we have identified and characterized a Schistosoma japonicum frizzled gene (Sjfz7). We found that Sjfz7 encodes a 698 amino acid protein with typical characteristics of Frizzled proteins. The immunohistochemical localization pattern showed that Sjfz7 protein was extensively distributed in almost all tissues of S. japonicum, including subtegumental muscle cells, parenchymal cells, intestinal epithelial cells and male and female germ cells. This indicated that Sjfz7-mediated Wnt signaling might be associated with the development of musculature, intestinal tract and reproductive organs in schistosome. Comparing mRNA levels between frizzled family members showed that Sjfz7 mRNA was consistently higher in the developmental stages analyzed, suggesting that Sjfz7 may be responsible for more functional tasks than other frizzled family members. Comparing frizzled mRNA levels between not fully developed and normal worms suggested that Wnt signaling might be abnormal in not fully developed worms.

[Prediction for helper T cell epitopes and its application in vaccine development against parasite infection].

Cellular immunity plays an important role in defense against diseases, such as pathogenic infection, autoimmunity and tumor. With the progress of molecular immunology, mechanisms of T cellular immunity, and the T cell epitopes and functional genomics, studies on the prediction based on data-derived for T cell epitopes has been highlighted, and could be one of the useful tools for application in vaccine development. This review summarizes theory and methodology of prediction for helper T cell epitopes, and their application in vaccine development against parasites, and new research directions are also discussed.

[Application of reverse vaccinology in Schistosoma vaccine development: advances and prospects].

OBJECTIVE: Current advances in reverse vaccinology based on the principle of "sequence-structure-function" and such integrated platform technologies as immunoinformatics, computer-aid design, and various high-throughput omics (including genomics, transcriptomics and proteomics) may pave a new way for the discovery of candidate vaccine molecules against schistosomiasis. Both theoretical prediction and experimental approaches conventionally used in the field of reverse vaccinology are briefly introduced in this review; and the applications of these approaches to screening and confirming candidate Schistosoma vaccine molecules are also summarized. Furthermore, potential research prospects of the application of reverse vaccinology to Schistosoma vaccine development are discussed by simulating immune effect mechanisms of immunization with radiation-attenuated cercaria vaccine in animal hosts and naturally acquired immunity in human population.

Characterization and expression pattern of a novel Frizzled 8 receptor gene in Schistosoma japonicum.

Wnt signaling as mediated by the Frizzled family receptors plays a vital role in the early development of animal embryos, organ formation, tissue regeneration and other physiological processes. In the present study, a novel Frizzled member, SjFz8, was isolated and characterized in Schistosoma japonicum. SjFz8 encodes an 1162-amino-acid protein with typical characteristics of Frizzled proteins. Quantitative real-time polymerase chain reaction analysis indicated that SjFz8 transcript level was highest in 7-day-old schistosomula. In adult stages, SjFz8 mRNA expression remained at a low level after male-female pairing. The immunohistochemical localization of the Fz8 protein revealed that it existed in almost all tissues of S. japonicum, including subtegumental muscle, parenchyma, oral suckers, ventral suckers, testes of the male and ovaries of the female. We speculated that the Wnt signaling pathway that was mediated by Fz8 might take part in regulating histogenesis and organogenesis during the schistosomulum period, and play an important role in regulating further growth and development of male and female worms.

SjCRT, a recombinant Schistosoma japonicum calreticulin, induces maturation of dendritic cells and a Th1-polarized immune response in mice.

BACKGROUND: It is well known that immunization of radiation-attenuated (RA) schistosoma cercariae or schistosomula can induce high levels of protective immunity against schistosoma cercariae reinfection in many animals. Many studies have shown that the Th1 cellular immune response is crucial for the protective effect elicited by RA schistosomula. However, the molecular mechanism of this strong protective immunity remains unclear. METHODS: The expression profiles of Schistosoma japonicum calreticulin (SjCRT) in RA and normal schistosoma-derived cells were investigated by flow cytometry. The effect of recombinant SjCRT (rSjCRT) on mouse dendritic cells (DCs) was determined by FACS, ELISA and RT-PCR analysis. We also analyzed the effects of SjCRT on the activation of spleen cells from mice immunized with rSjCRT by detecting lymphocyte proliferation and the cytokine profiles of splenocytes. RESULTS: We found that the expression level of SjCRT in the cells from RA larvae was significantly higher than that in cells from normal schistosomula at early stages of development (day 4). The results of effect of rSjCRT on mouse DCs showed that rSjCRT could induce phenotypic and functional maturation of DCs, and SjCRT bound to the surface of DCs through the CD91 receptor and could be engulfed by DCs. The results of activation of splenocytes from mice immunized with rSjCRT also demonstrate that rSjCRT can effectively stimulate the proliferative response of splenic lymphocytes, elicit splenocytes from immunized mice to secrete high levels of IFN-γ, TNF-α and IL-4, and activate CD4+ T cells to produce high levels of IFN-γ. CONCLUSION: SjCRT is one of the immunostimulatory molecules released from RA schistosomula cells, might play a crucial role in conferring a Th1-polarized immune response induced by RA cercariae/schistosomula in mice, and is a candidate molecule responsible for the high levels of protective immunity induced by RA schistosomula.