Sheila M. Lanham

Isolation of salivarían trypanosomes from man and other mammals using DEAE-cellulose.

Abstract Salivarian trypanosomes were separated from infected blood by adsorbing the particulate blood components on to DEAE-cellulose columns and eluting the trypanosomes. The optimal ionic strength of buffer was determined for a number of combinations of host-blood and parasite, and sufficient data were obtained to indicate the conditions for successful separation from untried combinations. By the technique, trypanosomes were recovered from lightly parasitemic and subpatent, as well as heavily parasitemic, infections; Trypanosoma brucei brucei alone was separated from T. vivax in a doubly infected blood. The specific adsorption-elution characteristics of the organisms with respect to DEAE-cellulose confirmed previous observations, cyclically transmitted isolations behaving similarly to those which were mechanically transmitted. The characteristics of T. simiae were unlike those of its fellow member of the subgenus Nannomonas, T. congolense, but like those of species in the subgenus Trypanozoon. Drug-resistant T. vivax tended to be more readily adsorbed than drug-sensitive T. vivax. The specific adsorption-elution characteristics of both trypanosomes and erythrocytes are interpreted in terms of their relative surface negative charge.

Influence of methods of preparation on the infectivity, agglutination, activity, and ultrastructure of bloodstream trypanosomes.

Contrary to the reports of others, the surface coat of Trypanosoma brucei brucei was not removed by extensive washing in the various media investigated; in fact, other than a deformed profile when washed in saline, ultrastructure was little affected by column separation and washing in any of these media. Washing in saline increased the agglutinability but reduced the activity and infectivity of the organisms. Washing in bicine-buffered saline-glucose did not impair activity or infectivity but increased agglutinability, albeit to a lesser extent than after washing in phosphate-buffered saline-glucose. The inclusion of 0.1% serum or plasma in the washing medium (phosphate-buffered saline-glucose or bicine-buffered saline-glucose) increased the activity of the T. b. brucei and did not increase agglutinability or impair infectivity. T. congolense and T. vivax were more susceptible to ionic strength changes than were T. b. brucei or T. lewisi, in that not only was their activity impaired, but they began to aggregate on standing in lower ionic strength buffers.

Some properties of the immunogens (protective antigens) of a single variant of Trypanosoma brucei brucei.

SUMMARY: Column-separated, clean trypanosomes were subjected to nitrogen cavitation and ultracentrifugation (90 min, 99000 g); the supernatant contained most of the immunogens but a few remained in the well washed residue. Column chromatography yielded a strongly immunogenic protein fraction (SAF1), small doses (0·035 mg protein/dose × 4) of which protected mice against a maximum challenge of 5 × 104 of the homologous variant organisms for a minimum of 17 weeks. No heterologous (variant or strain) protection was obtained with SAF1 either with increased doses or by homologous challenge followed by heterologous challenge 3 weeks later. Disc electrophoresis of SAF1 showed four anodic components intermediate in mobility between α-macroglobulin and transferrin of normal rat serum. Analytical ultracentrifugation indicated a substantial proportion of 6·5S protein together with 3·0S and 1–1·5S proteins. A smaller soluble antigen fraction (SAF8) consisted of 2·0S protein but preliminary experiment has not shown it to be immunogenic. Infected rat plasma separated after 6 h was as effective as SAF1 in protecting mice against homologous challenge whereas rapidly separated infected rat plasma gave only slight protection. Two identical precipitinogens were detected in infected rat plasma and SAF1 by immunodiffusion. Three additional precipitinogens were present in SAF1 and one other in the infected rat plasma. Antisera raised against various trypanosome fractions, including SAF1, agglutinated the homologous trypanosomes but to a lesser extent than antisera to living trypanosomes. Incubation of trypanosomes in either SAF1, or infected rat serum or plasma decreased their infectivity which was not altered by 8 h of incubation in normal rat plasma.