Fluorescent Antibody Analysis of Host Plasma Components on Bloodstream Forms of African Pathogenic Trypanosomes. I. Host Specificity and Time of Accretion in Trypanosoma congolense

Abstract

The presence, host specificity, and time of accretion of rat plasma components on the surface of Trypanosoma congolense were determined. Parasites collected at peak parasitemia (6to 10-day rat infections) were separated from blood cells on a DEAE-cellulose column and exogenous plasma was removed by washing. For immunoelectrophoretic analysis, the trypanosomes were lysed, the lysate centrifuged, and the soluble antigen reacted with rabbit antiserum developed against normal rat plasma. At least two antigen-antibody complexes were observed. To determine the location, host specificity, and time of appearance of the antigens shared by the host and parasite, a quantitative indirect fluorescent antibody method was used. The washed trypanosomes were fixed in 2.5% or 10% formalin before being subjected to the sandwich method. Formalin at a 2.5% concentration was the most suitable for the preservation of antigens. It was established by titrations that the largest difference in the fluorescence levels of the experimental and experimental control preparations occurred when the nonconjugated (first layer) rabbit antisera were used at 1:16 dilutions, and the fluorescein isothiocyanateconjugated anti-rabbit IgG serum (second layer) at a 1:40 dilution. Fluorescence differences between ratand mouse-derived trypanosomes stained with anti-rat plasma serum indicated that most, if not all, of the plasma components were host-specific. In vivo and in vitro experiments were performed to determine the length of time needed for appearance of new-host plasma components on the trypanosome surface. The rat-derived bloodstream forms when transferred to mice for 48 hr or incubated for 5 hr in normal mouse plasma emitted fluorescence quantitatively identical to that of trypanosomes grown in mice by serial passages. Incubation of mouse-derived trypanosomes in normal mouse plasma for 5 hr did not change their fluorescence measurements, but such trypanosomes incubated for 3 to 5 hr in normal rat plasma had fluorescence levels similar to those of rat-derived trypanosomes. The speed of acquisition and host specificity of the surface-bound antigens suggested that they were host plasma components accreted on the parasites. The occurrence of host plasma proteins on the surfaces of trypanosomes was suggested originally by Desowitz and Watson (1953) with regard to a rodent-adapted strain of Trypanosoma vivax. Subsequently, antigens common to trypanosomes and their hosts were reported from bloodstream forms of stercorarian and salivarian species on the bases of the results obtained by agglutination, double diffusion, immunoelectrophoresis, and direct fluorescent antibody methods. Among Stercoraria, such antigens were observed in Trypanosoma lewisi by D'Alesandro (1972) and Dwyer (1976). Among Salivaria, they were found in a mouse-adapted T. vivax (Ketteridge, 1970), Received for publication 8 December 1976. * This investigation was supported by Research Grant AI 00742-21, from NIAID, U.S. Public Health Service. t Predoctoral Trainee, supported by Training Grant 5 T01 AI 00226-13, from NIAID, U.S. Public Health Service. rat-adapted Trypanosoma brucei gambiense (Seed, 1974), and Trypanosoma brucei brucei (Vickerman, 1972; Le Ray, 1975). The present study was undertaken to ascertain if antigens resembling those of the host plasma occur on Trypanosoma (Nannomonas) congolense Broden. Since such host-parasite shared components were found, in vivo and in vitro experiments were designed to determine the length of time needed for the appearance of new host plasma components on the surface of the parasites. MATERIALS AND METHODS