Prevalence of Cryptosporidium spp. in pigs in Shanghai, China.

A survey on prevalence of Cryptosporidium spp. in pigs at 12 farms in 8 suburban districts and 1 county of Shanghai was conducted under Sheather's sucrose flotation protocol and modified acid-fast stain methods from 2006 to 2009. A total of 2323 faecal samples were collected and Cryptosporidium spp. oocysts were detected in 800 samples (34.4%). Cryptosporidium was found in all 12 pig farms. Significant variations of prevalence were observed in different farms ranging from 14.1% to 90.6%. A follow-up survey on a positive pig farm for 13 consecutive months revealed that the most serious infection of Cryptosporidium spp. in pigs happened in winter and spring, and the lowest infection season was summer. Cryptosporidium spp. infection was mainly found in piglets within 2 months and no infection was found among those pigs of 90-180 days of age. The genotype analyses were carried out through PCR-RFLP and partial sequences analysis of small subunit ribosomal RNA (SSU rRNA) in some of the positive samples. Cryptosporidium suis (57/69, 82.6%), Cryptosporidium pig genotype II (6/69, 8.7%) and mixed infection of above two species/genotype (6/69, 8.7%) were found to be the main species/genotype in pigs in Shanghai area.

In vitro and in vivo evaluation of small interference RNA-mediated gynaecophoral canal protein silencing in Schistosoma japonicum.

BACKGROUND: Schistosomiasis causes liver and intestinal damage and can be very debilitating. The pairing of a male worm with a female worm residing in the gynaecophoral canal of male plays a critical role in the development of female parasite. Because the male specific gynaecophoral canal protein of Schistosoma japonicum (SjGCP) is found in significant quantities in the adult female worm after pairing, it could play an important role in parasite pairing. METHODS: In the present study, three small interfering (si)RNA duplexes targeting the SjGCP gene were designed, synthesized and the silencing effects were evaluated in vitro as well as in mice infected with S. japonicum in vivo. RESULTS: In vitro studies using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR revealed the reduction of SjGCP at the transcript level. Similarly, western blotting and immunofluorescence studies showed its reduction at the protein level after treatment of parasites with siRNAs. At a concentration of 200 nM, two siRNAs totally abolished the parasite pairing. To evaluate such a pairing inhibitory effect in vivo, mice infected with S. japonicum were treated with siRNA and both parasite pairing and burden were evaluated. In vivo tests confirmed the in vitro silencing effect of SjGCP siRNA and revealed that the systemic delivery of siRNA significantly inhibited early parasite pairing and the associated burden. CONCLUSIONS: Our preliminary results demonstrated that the SjGCP plays an important role in pairing and subsequent development in S. japonicum, and its silencing might have potential as a therapeutic approach for controlling schistosomiasis.

[Construction and expression of recombinant baculovirus with Schistosoma japonicum SjPP gene].

OBJECTIVE: To express the soluble recombinant Schistosoma japonicum SjPP proteins in TN5B1-4 cells. METHODS: The total RNA was extracted from adult worms of Schistosoma japonicum. The whole coding sequence of SjPP gene was synthesized by RT-PCR and cloned into donor plasmid. The recombinant donor pFastBac-SjPP was transformed into E.coli DH10Bac forming Bacmid-SjPP which was transfected into insect cell with cational lipofectin. The fusion protein SjPP was analyzed with SDS-PAGE and Western blotting. RESULTS: The infective recombinant baculovirus Bacmid-SjPP was obtained and SjPP protein was expressed in insect cells. CONCLUSION: The recombinant protein SjPP has been expressed in insect TN5B1-4 cells with proper antigenicity.

[Cloning, expression and characterization of a gene encoding signal transduction protein Wnt4 of Schistosoma japonicum].

Wnt proteins together with their downstream effectors forms a set of important signal pathways. The Wnt signal pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis. Wnt4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. With RACE technique based on a EST identified in our lab, a novel gene including a complete open reading frame was cloned and named Sjwnt4 (GenBank accession No. DQ643829). Sequence analyses showed that SjWnt4 had a typical characteristics of Wnt family proteins, sharing 43% similarity to Dugesia japonica and 37% to human Wnt4. The ORF of Sjwnt4 contains 1311 nucleotides, encoding 436 amino acid with 49.6 kD molecular weight. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of Sjwnt4 is highest in the 19 days schistosomula, followed by 44 days female worms, 14 days schistosomula, 31 days adult worms and 44 days male worms, suggesting a stage-and-gender differential express. The Sjwnt4 cDNA fragment was subcloned into a modified expression vector pGEX-4T-2 and transformed into E. coli BL21 (DE3) cells, and the production of recombinant Sjwnt4 protein fused to a GST tag was analysed. In the presence of IPTG, the 76kD fusion protein was expressed in included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum specific to Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the regulation mechanism of the Wnt signaling pathway during the development especially gonadal differentiation processes of Schistosoma japonicum.

[Construction of phage display cDNA library from adult worms of Schistosoma japonicum].

OBJECTIVE: To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. METHODS: Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. RESULT: Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library. CONCLUSION: The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

[Studies on phage displayed mimotopes of a protective monoclonal antibody (SSj14) against Schistosoma japonicum].

To obtain peptides mimicking epitope of a protective McAb SSjl4 specific to Schistosoma japonicum and investigate their immuno-protection effects. A phage random 12 peptide library was screened using purified McAb SSj14, 33 clones were picked up for specificity identification by ELISA. The epitope of each positive clones were detected by the sequencing analysis technique. The antigenicity of three positive clones (P1, P2 and P11) and their mixture cock-tail were further confirmed by Western-blotting, and their protective efficiency were evaluated by mice vaccination experiment. IL-12 level between the vaccinated mice and control mice were compared. 30 positive phage clones were obtained, which represented 11 different epitopes respectively, there were a similar sequence "H-N/Q-X-S-P/F-X-X-L-A-T" among all of the epitopes. Western-blotting showed that all of the three tested clones were recognized by McAb SSj14. Significant adult worm reduction (13.84% to approximately 52.83%), liver tissue egg reduction (34.17% to approximately 65.47%) as well as fecal egg reduction (28.89% to approximately 73.78%) were observed in mice vaccinated with phages of P1, P2, P11 and mixture of three clones when compared with those of the blank control group, among them, the mice vaccinated with the mixture of phage clones got higher protection than any of the mice injected with only one kind of clone phages. At the same time, the IL-12 level in serum of vaccinated mice was found higher than those of the blank control one, this suggest that IL-12 may correlate with the protective efficiency induced by the clone phages. The study provides a new way for developing an effective vaccine against S. japonicum.

Dose-dependent inhibition of gynecophoral canal protein gene expression in vitro in the schistosome (Schistosoma japonicum) by RNA interference.

The gynecophoral canal protein gene SjGCP of Schistosoma japonicum that is necessary for the pairing between the male and female worms is specifically expressed in the adult male worm. This protein is widely distributed in the adult female worm after pairing. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence were employed to analyze the relationship between the RNAi effect and dsRNA dosage in the parasites. The results revealed that the inhibition of SjGCP expression by siRNA is dose-dependent. RT-PCR analysis showed that the SjGCP transcript level was reduced by 75% when 100 nM dsRNA was applied.

Proteomic analysis of differentially expressed proteins between the male and female worm of Schistosoma japonicum after pairing.

Identification of differentially expressed proteins between the male and female worm of Schistosoma japonicum may provide new insights into the development of schistosomes, especially the molecular mechanism of female worm maturation induced by the male worm after pairing. Comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry were employed to separate and identify differentially expressed proteins between the male and female worm after pairing. Soluble and hydrophobic proteins from egg, schistosomulum (14 days), and female and male worms at adult stage (42 days) were separated by a sequential extraction method followed by 2-DE and 2-DE images were constructed. There were 1016 +/- 67, 1808 +/- 89, 1142 +/- 45 and 1288 +/- 32 spots detected for soluble proteins and 1425 +/- 108, 952 +/- 59, 847 +/- 75 and 965 +/- 69 spots for hydrophobic proteins from egg, schistosomulum, and adult stage female and male worms, respectively. The differentially and uniquely expressed proteins from male and female worms after pairing (42 days) include 41 +/- 4 and 23 +/- 2 unique spots for soluble proteins, and 11 +/- 1 and 26 +/- 3 unique spots for hydrophobic proteins, respectively. Matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry were employed to analyze 12 unique spots from the female worm and 16 unique spots from the male worm for peptide mass fingerprinting and sequencing. The results showed that the main functions of these differentially expressed proteins were in signal transduction, metabolism and transcriptional regulation etc. Comparison of the schistosomes proteome between male and female worms may permit the identification of protein candidates for the development of vaccines or new targets for drug development against schistosomiasis.

[Cloning, expressing and functional analysis of SjMF4, a novel Schistosoma japonicum gene].

Based on the phenomenon of the natural anti-schistosomiasis in Microtus fortis, the sera from normal Microtus fortis were employed to immunoscreen the cDNA library of adult Schistosoma japonicum (Chinese strain), two positive clones were obtained, RACE technique was further applied to amplify one of the clones, and a cDNA fragment with an ORF was identified. Sequencing revealed that it was a novel gene of Schistosoma japonicum, and it was named SjMF4 (Schistosoma japonicum Microtus fortis 4). Then the structure and functional motifs of SjMF4 were analysed. The gene was subcloned into pET-28a(+) vector; the recombinant protein showed good antigenicity in Western blotting. The gene was further subcloned into eukaryotic expression vector pcDNA3 to construct the DNA vaccine containing SjMF4. Immune experiments in mice showed significant protection that the recombinant plasmid did induce 28.64%+/-3.82% worm reduction and 21.73%+/-3.98% egg reduction than controls against the Schistosoma japonicum cercaria challenge.

[Protective effect induced by vaccination with a partial cDNA expression library of Schistosoma japonicum in mice].

The cDNA fragments of interest were amplified using Sj lambda ZipLox library as the templates by PCR and then cloned into a eukaryotic expression vector p-CMV-GH; A small number of DNA fragments inserted in the recombinants was identified by restriction cleavage, EST sequencing and bioinformatical analysis; mice were injected intramuscularly with the expression library (L-CMV-SjR) or sublibraries(L-CMV-SjR1, L-CMV-SjR2 and L-CMV-SjR3), immunized mice were challenged with Schistosoma japonicum cercariae on day 35, the levels of IgG antibodies in sera from the immunized mice were detected by ELISA. The results demonstrated that a partial cDNA expression library of S.j, with approximately 10(5) transformants, was constructed, most of the recombinants contained the insert DNA fragments of interest, and these fragments had the features of protein-coding sequences for Schistosome. There were no significant differences for the levels of IgG antibodies in sera from all of the immunized groups. Mice immunized with L-CMV-SjR, L-CMV-SjR1 and L-CMV-SjR2 developed significant protective effect against Sj infection compared to control mice injected with the empty plasmid, the rate of worm reduction was about 30%.

[Cloning and expression of 21.7 kD protein gene of Schistosoma japonicum (Chinese strain)].

A 558 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with a pair of primers that were designed according to published Sj21.7p gene encoding 21.7 kD protein of Schistosoma japonicum(Philippines strain). Sequence analysis indicated that this frame, named Sj21.7 (Ch), with 99% homology to Sj21.7 p, contained a complete open reading fragment (ORF) of 21.7 kD protein gene of Schistosoma japonicum(Chinese strain). The amino acid sequence shared 98% homology with 21.7 kD protein of Schistosoma japonicum. This fragment was cloned into the expression vector pET28a (+) and subsequently expressed in Escherichia coli with IPTG induction. SDS-PAGE analysis revealed that the molecular weight of this expressed product was 25.4 kD. Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.

Cloning of Schistosoma japonicum Chinese Strain TPI Gene and Characterization of Its Expression Product in Escherichia coli.

Triose-phosphate isomerase is an important candidate for schistosoma antigens. An 800 bp DNA fragment was amplified by RT-PCR from adult Schistosoma japonicum mRNA. Sequence analysis revealed that this fragment contained S. japonicum (Chinese strain) triose-phosphate isomerase gene. Then this gene was cloned into the expression vector pGEX-4T and subsequently expressed in E. coli. The recombinant GST-fusion protein was purified by glutathione agarose affinity chromatography. Its molecular weight was determined to be 54 kD. The yield of expression was around 30 mg/L E. coli culture. Western blotting showed that the recombinant protein had good antigenicity which could be helpful for the making of anti-S. japonicum multi-valent recombinant vaccine.

Cloning of Schistosoma japonicum Chinese Strain Fatty Acid Binding Protein (Sj-FABPc) Gene and Its Overproduction in Escherichia coli.

A 600 bp DNA fragment was amplified by PCR, from an adult Schistosoma japonicum cDNA library. Sequence analysis revealed that this fragment containedthe S. japonicum Chinese Mainland strain fatty acid binding protein (Sj-14FABPc) gene. This gene was then cloned into the expression vector pGEX-2T, and subsequently expressed in Escherichia coli. The recombinant GST-fusion protein could be purified by glutathione agarose affinity chromatography. Its molecular weight was about 41 kD. The yield of expression was around 25 mg/L E. coli culture. The immunological test suggested that the recombinant protein had good antigenicity, and could be developed into a new vaccine molecule of S. japonicum.

Studies on the Expression of the 28 kD Glutathione S-transferase Gene from Schistosoma japonicum in the Silkworm (Bombyx mori) Larvae and Insect Cells.

The gene of the 28 kD glutathione S-transferase (GST) from the Chinese strain of Schistosoma japonicum had been expressed in the silkworm (Bombyx mori) cells and larvae by Bombyx mori nuclear polyhedrosis virus (BmNPV) vector which had been modified. The GST gene was inserted into the right position of the BmNPV genome as identified by Southern hybridization. The product was a 28 kD protein, had GST activity and antigenicity. The yield in BmN cells (1x10(6) cells/ml) was 0.77 mg and more than 5 mg in a silkworm larvae. All the results showed that it is possible to develop the GST expressed in the silkworm larvae to a new kind of genetic engineered schistosomiasis vaccine.