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

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Abstract
The presence, host specificity and time of accretion of rat plasma components on the surface of T. congolense were determined. Parasites collected at peak parasitemia (6- to 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 2 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 2.5% concentration was the most suitable for the preservation of antigens. Titrations established that the largest difference in the fluorescence levels of the experimental and experimental control preparations occurred when the nonconjugated (1st layer) rabbit antisera were used at 1:16 dilutions, and the fluorescein isothiocyanate-conjugated anti-rabbit Ig[immunoglobulin]G serum (2nd layer) at a 1:40 dilution. Fluorescence differences between rat- and 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 h or incubated for 5 h 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 h did not change their fluorescence measurements, but such trypanosomes incubated for 3-5 h 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.

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