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.

The ultrastructure of the gravid uterus of Brugia pahangi, another rich source of antigen of the filarial parasite.

The gravid uterus with zygotes and microfilariae in utero of Brugia pahangi, a rich source of antigen as revealed by a recent immunofluorescent technique, were studied ultrastructurally. The epithelial cells of uterus show ultrastructural features of synthetically active cells. Their secretions may provide nutrients for the egg in utero. On the basal side, the uterine epithelial cells may also secrete substances to form the basal lamina of the uterus which is rather thick and irregularly fused with the basal lamina lining the body wall where the pseudocoelomic cavity is obliterated. For the most part, the uterine basal lamina contains uniform granular material of moderate electron density. There are also elongated visceral muscle cells embeded in it, and which surround the uterus, with adjacent cells overlapping. The gravid uterus contains several stages of developing microfilariae within its lumen, the cleaving zygotes are also present at another level. The morula of zygotes are composed of several closely packed cells surrounded loosely by their own egg shell membranes. The egg shell becomes more convoluted as development proceeds. The egg shell surrounding the developing microfilariae in utero is secreted by the uterine epithelium. This structure later becomes the sheath of circulating microfilariae, and is highly antigenic as indicated by intense labeling with fluorescent antibodies.

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.

The possible antigenic sources of brugia pahangi.

The antigenic sources of adult and the third larval (L3) stages of Brugia pahangi were detected by indirect immunofluorescent technique. Six panels of antisera were used, including human antisera against Brugia malayi and Wuchereria bancrofti, cat antisera against B.malayi and B.pahangi and jird antisera against B.malayi and B.pahangi as primary antibodies. All antisera gave the same results, although four of the six were not infected by B.pahangi. This indicates non-species specificity, and B.pahangi, B.malayi and W.bancrofti must share most of the common antigenic molecules. All antisera reacted well with the surface of L3 B.pahangi in the whole mount preparation. This indicates non-stage specificity. The most intense fluorescence was located at the epicuticle, the basal lamina lining the body wall, the gut and the reproductive tract, the egg shell in utero and the sperm. The hypodermis, the muscle cells, the cuticle beneath the epicuticle, the epithelial cells of the gut and the reproductive tract showed moderate fluorescence. The least fluorescence was observed in the egg interior.

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.