Cloning, characterization, and expression of vitelline protein BI and its encoding gene in the liver fluke, Fasciola gigantica.

A cDNA containing a 813 bp open reading frame encoding vitelline protein BI (FgVPBI) of Fasciola gigantica was cloned. FgVPBI has 96% sequence identity with VPBI of Fasciola hepatica and 84% identity with VPBII F. hepatica. It is far less similar to eggshell precursor proteins of other trematode species, for example, 29% identity with C. sinensis. Northern blot hybridization of total RNA from adult parasites demonstrated a FgVPBI transcript with a size of 1,000 nucleotides. FgVPBI mRNA is localized in the vitelline cells in both vitelline glands and intrauterine eggs. Recombinant FgVPBI was expressed as a 31.5 kDa protein in Escherichia coli and used for production of a polyclonal antiserum in rabbits. The FgVPBI antiserum detected immunoblotted rFgVPBI and native eggshell precursor protein at molecular weights of 31.5 kDa and 31 kDa, respectively. Immunolocalization showed strong staining in the cytoplasm of vitelline cells, in eggshell globules and the shells of eggs.

Molecular cloning and characterization of a glutathione S-transferase encoding gene from Opisthorchis viverrini.

An adult stage Opisthorchis viverrini cDNA library was constructed and screened for abundant transcripts. One of the isolated cDNAs was found by sequence comparison to encode a glutathione S-transferase (GST) and was further analyzed for RNA expression, encoded protein function, tissue distribution and cross-reactivity of the encoded protein with other trematode protein counterparts. The cDNA has a size of 893 bp and encodes a GST of 213 amino acids length (OV28GST). The most closely-related GST of OV28GST among those published for trematodes is a 28 kDa GST of Clonorchis sinensis as shown by multiple sequence alignment and phylogenetic analysis. Northern analysis of total RNA with a gene-specific probe revealed a 900 nucleotide OV28GST transcriptional product in the adult parasite. Through RNA in situ hybridization OV28GST RNA was detected in the parenchymal cells of adult parasites. This result was confirmed by immunolocalization of OV28GST with an antiserum generated in a mouse against bacterially-produced recombinant OV28GST. Both, purified recombinant and purified native OV28GST were resolved as 28 kDa proteins by SDS-PAGE. Using the anti-recOV28GST antiserum, no or only weak cross-reactivity was observed in an immunoblot of crude worm extracts against the GSTs of Schistosoma mansoni, S. japonicum, S. mekongi, Eurytrema spp. and Fasciola gigantica. The enzyme activity of the purified recombinant OV28GST was verified by a standard 1-chloro-2, 4-dinitrobenzene (CDNB) based activity assay. The present results of our molecular analysis of OV28GST should be helpful in the ongoing development of diagnostic applications for opisthorchiasis viverrini.

Production and characterization of a monoclonal antibody against recombinant glutathione S-transferase (GST) of Fasciola gigantica.

A monoclonal antibody (MoAb) against a recombinant glutathione S-transferase (rGST) of F. gigantica was produced in BALB/c mice. Reactivity and specificity of this monoclonal antibody was assessed by ELISA and immunoblotting. Six stable clones, namely 3A3, 3B2, 3C6, 4A6, 4B1 and 4D6 were obtained, All these MoAb reacted with rGST and native GST at a molecular weight of 28 kDa and found to be IgG1, kappa-light chain isotypes. These MoAb cross-reacted with Schistosoma mansoni and Schistosoma japonicum antigens at molecular weights of 28 and 26 kDa, respectively, but no cross-reactions were detected with antigens of Eurytrema and Paramphistomum spp. The localization of GST in metacercaria, 7-week-old juvenile and adult F. gigantica was performed by immunofluorescence technique, using MoAb as well as polyclonal antibody (PoAb) to the native protein as probes. In general, all clones of MoAb gave similar results and the pattern was quite similar to staining by PoAb. The fluorescence was intense, which implied the presence of a high concentration of GST in the parenchymal tissue in all stages of the parasite. However, the parenchymal cells were not evenly stained which implied the existence of subpopulations of this cell type with regard to GST production and storage. In addition, in adult and juvenile stages a moderate fluorescence was present in the basal layer of the tegument, while light fluorescence was observed in the caecal epithelium, cells in the ovary, testis and vitelline gland of the adult. In the metacercaria stage, in addition to parenchymal tissue, the tegument and tegumental cells were stained relatively more intense with MoAb and PoAb than in other stages.

Low cost pregnancy test and HCG stability.

A modified technique for pregnancy slide test (Roche) using hypodermic needles No.23 as droppers for urine and needles No.25 as droppers for antiserum and for human chorionic gonadotropin (HCH)-latex suspension was reported. One hundred positive-and 26 negative-urine specimens by regular Roche pregnancy slide test were used as study samples. The results showed that the modified technique was compatible with the standard method regarding sensitivity and reliability. The modified technique, is however, 6.76 times less expensive than the standard method. The authors recommend this new technique to be used in hospital laboratories and clinics as routine laboratory test. In this study if was also found that the urine specimens that kept at room temperature for up to one year the HCG hormone remained active though the number of positively was reduced if kept 1ฝ year and 2 years to 98.41 and 93.65 percent respectively. Urine specimens that preserved in refrigerator freezer remained positive for HCG hormone for up to 2 years.