Date of Award

1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Abstract

Monoclonal antibodies (mAbs), raised against whole, fixed, uncoated, culture forms of Trypanosoma (Duttonella) vivax, were used to identify two invariant membrane proteins of this protozoan parasite. Since non-variant membrane proteins of the cell surface, flagellar pocket and endocytic pathway are potential targets for the control of trypanosomiasis of livestock by immunization, the identification and characterization of invariant membrane proteins is a necessary preliminary step.;A 65 kDA invariant membrane glycoprotein (gp65), identified using mAb 4E1, was the main focus of this study. Immunolocalization studies using the monoclonal antibody (mAb 4E1) for immunofluorescence staining and immunoelectron microscopy, demonstrated that the 65 kDa antigen was associated with tubulo-vesicular profiles in the posterior region of the bloodstream form parasite. Endocytosis and co-localization experiments revealed that gp65 was associated with an endocytic compartment of T. vivax which is morphologically and temporally similar to the endosomal system of mammalian cells. Double labelling experiments using the mAb and a polyclonal anti-variant surface glycoprotein antibody (R{dollar}{lcub}\alpha{rcub}{dollar}VSG) to simultaneously localize both gp65 and intracellular VSG, demonstrated that there was little overlap in the distribution of these antigens. Thus, gp65 is associated with tubules and vesicles that are involved in endocytosis but which appear to be distinct from VSG processing pathways in the cell.;A 35 kDa T. vivax antigen was shown in immunolocalization studies to be associated primarily with the surface of bloodstream forms of the parasite. Although T. vivax 3{dollar}\sp{lcub}\prime{rcub}{dollar}-nucleotidase activity, a surface membrane enzyme in other trypanosomatids, migrated at 35 kDa on SDS-PAGE gels, it is doubtful that the 35 kDa antigen identified with the monoclonal antibody (mAb 4B11) is specific for the T. vivax 3{dollar}\sp\prime{dollar}-nucleotidase since the two proteins exhibited different capacities to bind to immobilized Concanavalin A.;Both T. vivax invariant antigens have potential as targets for disease control based on their location in the cell and thus merit further study to this end. In addition, gp65 is the first putative marker for an endosomal compartment of trypanosomes and has potential for use in the further study of endocytosis in African trypanosomes, a process upon which these parasites are dependent upon for survival.

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