Classification of the parenchymal cells in Fasciola gigantica based on ultrastructure and their expression of fatty acid binding proteins (FABPs).

Parenchymal cells in adult Fasciola gigantica can be classified into three types based on their ultrastructural features and different quantities of fatty acid binding protein (FABP) being stored. Parenchymal cell type 1 (Pc1) has pale cytoplasm consisting largely of a loose network of fine fibers, and it contains few mitochondria but numerous glycogen particles. This cell type may be specialized in the storage and metabolism of glycogen and glucose. Parenchymal cell type 2 (Pc2) has similar cytoplasmic features as Pc1 but contains more numerous mitochondria, and high concentration of FABP as reflected by high density of immunostaining and immunogold labeling using specific monoclonal antibody (MoAb) to FABP as probe. Pc2 may, thus, specialize in the storage and metabolism of fatty acids and other lipids. Parenchymal cell type 3 (Pc3) has dense cytoplasm containing large amount of rough endoplasmic reticulum, Golgi complex and mitochondria, which is typical of a secretory cell. Furthermore, Pc3 has very little glycogen particles and is not stained by MoAb against FABP. It could, thus, be concerned with the synthesis of fibers, which form the scaffold of the parenchyma.

Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica.

A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hybridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F. gigantica. This MoAb was found to be of the isotype IgG(1), kappa-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion-secretion material of the adult, whole-body extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7-week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development.

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.