The infectivity of single flagellates of Trypanosoma cruzi

Abstract

S in gle organism s o f Trypanosoma cruzi o f the virulent Peru strain were isolated b y direct visualisation a n d were injected peritoneally in to C F I mice. Sin gle trypanom astigotes o f different m o rp h o lo g y a n d from different sou rces (m ouse ifficulties attendant o n the p erform ance o f the cio n in g technique are d iscu sse d a n d som e in d ication is given o f h ow these p roblem s can be overcome. IN T R O D U C T IO NUsing a Peru strain of T ryp an oso m a cru zi it has been shown that the number of trypanomastigotes inoculated infiuences the course of infection in the mouse host as regards survival. Also that one organism of this strain obtained by serial dilution could induce a patent, often lethal, infection in a mouse6. Since such serial dilution may be attended by ináccuracies4 it was decided to directly visualise and inoculateone flagellate in a further series of experiments to study the infective potential of individual flagellates of this strain. M A T E R IA L A N D M E T H O D SThe virulent Peru strain of T. cru z i9 was oontinuously maintained in the laboratory by weekly syringe passage in C FI mice, in liquid culture medium (brian heart infusion broth wíth 10% human blood) and in R h o dnius p rolixu s. Suitably diluted suspensions of flagellates from these sources were prepared in a diluent of heparinised (5units/ml) normal saline pH 6. Using a fine pipette small drops of suspension (1-2mm diameter) were dropped onto clean microscope slides and scanned at a magnification of 500 X. If oníy one flagellate was seen this was confirmed by a second observer. Epimastigotes and trypanomastigotes can be distinguished in isolates ^rom bug faeces and culture. Trypanomastigotes are characterised by a posterior terminal kinetoplast and a rapid flickering movement across the field. Epi mastigotes are broader, without a terminal kinetoplast and lessactivewithclearlydefined serpiginous movements of the flagellum. A distinction was made between broad and narrow blood trypanomastigotes. Drops where only one organism was visualised were drawn into a 1 ml plastic syringe already charged with 0.1 ml of normal saline through a fine needle. A different syringe and needle was used for each experiment. The syringe contents were inoculated intra peritoneally into a 9 to 12gm mate CFI mouse. Only fully motile flagellates were inoculated.