SummaryClones of animal-infective bloodstream forms of Trypanosoma brucei (stocks S.427 and LUMP 227) were made by transferring a single organism from bloodstream-form cultures into each well of Microtest II Tissue Culture Plates containing bovine fibroblast-like feeder cells. When the number of trypanosomes increased to 102–103/well on days 4–16, they were transferred into plastic T-25 culture flasks also containing feeder cells and fresh medium. Cultures were thereafter maintained by partially replacing the trypanosome suspension with the same volume of fresh medium (diluting the density to 2–5 × 105 trypanosomes/ml) every 24 h. Sub-cultivations could be made by transferring 1–2 ml of the trypanosome suspension to a new culture flask at 4–5 day intervals. A total of 42 clones in the 3 series TC221, TC52 and TC227, carrying variable antigen types (VATs) 221, 052 and ILTat 1·4, respectively, have been established. Average population doubling times for clones of TC221, TC52 and TC227 were 8·7, 14·5 and 15·5 h respectively. Of 35 populations examined, 34 clones retained the original specificity of their VATs for at least 8–32 days from cloning. One cloned population of TC52 consisted of 99·8% VAT 052 and 0·2% VAT 221 at the time when the first VAT test was made on day 18.

STJMMAEYEighteen clones of variable antigen type 052 of Trypanosoma brucei stock S. 427 were derived and maintained as animal-infective bloodstream forms in vitro for up to 60 days of cultivation. The antigenic composition of such clones was monitored weekly by immunofluorescent analysis of viable trypanosomes, using antisera raised to isolated variant-specific surface glycoproteins of both 052 and a variable antigen type (221) which consistently appeared in the first relapse population of type 052 in vitro. The appearance of new variants was detected in 9 of the 18 clones 18-46 days following initiation of the clone and variable antigen type 221 was found in all 9 clones. On one or more occasions in 8 of such clones, viable trypanosomes were found which did not react with either antiserum but were mouse-infective on the 4 occasions tested and probably represent other variable antigen types. The process of antigen, variation in vitro appears to resemble the process in vivo except that new variant types are detected earlier in vivo. This possibly results from different growth rates of the trypanosomes in vivo and in vitro, together with the fact that elimination of the initial variant population by the host's immune response facilitates the detection of newly arising variable antigen types in vivo.

Trypanosoma brucei grew in the presence of bovine fibroblast-like cells in Hepes-buffered RPMI 1640 medium with 20 percent fetal bovine serum for more than 220 days at 37 degrees C. The organisms grown in this system were infective to mammalian hosts, retained the morphological and biochemical characteristics of long slender bloodstream forms, and displayed variant-antigen on their surfaces.

We have described some rearrangements of a variable-antigen gene in T. brucei. We suggest that there are two copies of the ILTat 1.2 variable-antigen gene in each of a number of trypanosome clones closely related by sequential relapses. Both copies of the gene undergo rearrangements, apparently the result of insertion and deletion of lengths of DNA in a region at or beyond the 3' end of the coding sequence, giving rise to different-size restriction enzyme fragments in different clones of trypanosomes. No feature of these rearrangements can be correlated with expression of the gene. Our data differ from those of Hoeijmakers et al. (1980) in two important respects: (1) Neither copy of the gene remains in a constant genomic context in all trypanosome clones. (2) We do not see an extra copy associated with the expression of the gene. These observations do not suggest any obvious mechanism for the phenomenon of antigenic variation.

The TP regimen's increased mean survival cost per YLG (inpatient and outpatient settings) adds a substantial benefit at an acceptable cost compared with CP therapy.