The present research provides the first evidence for specific receptor binding of polyhedra-derived baculovirus (PDV) to host cells and to lepidopteran brush border membrane vesicles (BBMV) and demonstration of entry via a non-endocytotic pathway involving direct membrane fusion. The technique of fluorescence-activated cell sorting (FACS) analysis was employed to investigate the specificity of binding between the PDV phenotype of Lymantria dispar multicapsid nuclear polyhedrosis virus (LdMNPV) and host membranes. Fluorescein-labeled PDV was found to bind in a saturable manner to the gypsy moth cell line, IPLB-LdEIta, and to L. dispar BBMV. Excess levels of unlabeled PDV were highly efficient in competing with FITC-labeled PDV for limited receptor sites further supporting the specificity of the interaction. Major reductions in virus binding (as high as 70%) after protease treatment of cells indicated that a protein receptor is involved. A fluorescence dequenching assay of membrane fusion with octadecyl rhodamine B (R18)-labeled PDV as used to identify PDV fusion to host cells and BBMV. Fusion of PDV occurred at 27$\sp\circ$C to both target membranes, as well as at 4$\sp\circ$C at approximately 55% of the levels achieved at 27$\sp\circ$C. Virus fusion to BBMV occurred throughout the pH range of 4-11 with dramatically increased fusion levels (3x) under the alkaline conditions normal for lepidopteran larval midguts. Treatment of tissue cells with chloroquine, a lysosomotropic agent, did not significantly affect PDV fusion to cells or infectivity in tissue culture. To begin to characterize viral and host proteins involved in establishment of infection, monoclonal antibodies (MAbs) were produced against LdMNPV PDV as well as L. dispar BBMV. While no PDV-neutralizing MAbs were isolated, anti-BBMV MAbs were identified which appeared to alter either PDV fusion or infectivity. To determine whether LdMNPV occlusion bodies (OBs) may contain a soluble enhancing factor similar to that identified for several granulosis viruses, soluble LdMNPV OB proteins were tested in larval bioassays for enhancement of PDV infectivity and assayed for the ability to degrade L. dispar peritrophic membrane proteins. Soluble LdMNPV OB proteins were unable to enhance LdMNPV infectivity and did not degrade larval PM proteins.

Comparative cytological and molecular phylogenetic data suggest that amitochondriate trichomonads (Archaeprotista: Parabasalia) are an early branching lineage of eukaryotes. Prominent in their cells is the karyomastigont, a cytoskeletal organellar system that includes a nucleus, 4–5 kinetosomes, an axostyle, a parabasal body (Golgi complex), and associated fibers. The karyomastigont is an ancestral character of eukaryotes. Genera of the trichomonad family Calonymphidae (Calonympha, Coronympha, Metacoronympha, Stephanonympha and Snyderella) are polymastigont: multinucleate cells with many karyo- or akaryomastigonts (karyomastigonts that lack nuclei). Termite hindgut trichomonads were examined for their susceptibility to antibiotics, pattern of cell division, and to test the hypothesis that DNA is associated with centriole-kinetosomes. Several types of bacterial symbionts are harbored on the surface membranes, in the cytoplasm and nuclei of these anaerobic protists rendering them susceptible to antibacterial antibiotics. Caduceia sp., a devescovinid trichomonad with four types of bacterial symbionts, was removed within 14 days from the Cryptotermes cavifrons (Florida) gut when penicillin and streptomycin were fed to the termite. Snyderella tabogae was far less susceptible to the antibiotic treatment, which also enhanced formation of calcium-rich crystals in the intestine. Metacoronympha divides asymmetrically. M. senta from Incisitermes nr. incisus (Trinidad) also showed a bimodal distribution of cell size. Approximately one third of the population averaged 100 μm in length with as many as 1000 karyomastigonts, unlike the previously described 50 μm mean length with up to 350 karyomastigonts. The cytoskeleton of Snyderella tabogae, the only calonymphid whose complete set of nuclei are not attached to centriole-kinetosomes, consists solely of 500 or more akaryomastigonts (>2000 undulipodia and their kinetosomes). In Snyderella tabogae, groups of 20–50 akaryomastigonts simultaneously beat and form locally organized regions of the cortex. These polymastigont cells are ideal to seek centriole-kinetosome-associated DNA. Fluorescent cytological (DAPI, SYTOX, acridine orange, ethidium bromide, propidium iodide) as well as Feulgen stains were negative: no DNA was present in the centriole-kinetosomes of S. tabogae. However, DAPI accumulates in the parabasal bodies (Golgi complex).

The first part of this study examines vaccine candidates for African pathogenic trypanosomes. Nonvariant antigens (NVAs) limited to bloodstream form stages of Trypanosoma congolense, have been identified using immunodiffusion, immunoelectrophoresis, and Western blotting techniques. Results from experiments employing surface biotinylation, iodination, and immunogold labeling suggest that these NVAs are exposed on the parasite surface and thus, if appropriately presented might afford the mammalian host a protective immunity. Trypanosoma brucei brucei development in Glossina provides another opportunity to block parasite development via passive immunization against procyclic forms. Results obtained from studies designed to evaluate this possibility suggest that ingested anti-procyclic antibody had little influence on parasite development within Glossina. The apparent resistance of antibody to enzymatic degradation for up to 120 h postingestion, as determined by Western blotting, suggests that antibody inactivation, rather than destruction is responsible for these results and provides a rationale for future investigations. The second part of this study investigates virulence mechanisms and identifies possible diagnostic antigens of the human urogenital protozoan parasite Trichomonas vaginalis. Plasma membrane isolated from T. vaginalis strains differing in virulence contain qualitative but not quantitative differences in their protein composition as determined with the aid of Western blotting. Two proteins in the 150 kDa range appeared more abundant in mild strains, while proteins of 43 and 25 kDa appeared more abundant in the membranes of virulent strains. These latter proteins are apparently heavily glycosylated as indicated by their ability to bind ConA and SBA lectins. Possibly, these glycoproteins may contribute to virulence mechanisms of the parasite. In attempts to develop a reliable diagnostic assay, soluble antigens shed by T. vaginalis were identified using Western blotting techniques. Five soluble antigens were initially identified in supernatant fluids obtained from parasites grown on TYM medium supplemented with 1% normal human plasma (NHP). Four of these proteins immunologically cross-reacted with NHP and are thus of limited diagnostic potential. A 100 kDa antigen, lacking in NHP cross-reactivity, however, could be isolated from all parasite strains examined and maybe of some use in developing a diagnostic assay.