A high level of mixed Trypanosoma brucei infections in tsetse flies detected by three hypervariable minisatellites

The issue of whether genetic exchange occurs at a significant frequency in natural populations of Trypanosoma brucei is controversial and one of the a rguments against a high frequency has been the apparent lack of host infect ions with mixtures of trypanosome genotypes. Three minisatellite markers (M S42, CRAM, 292) within the coding regions of three genes have been identifi ed and PCR based methods developed for detecting Variation at these loci us ing crude lysates of infected blood as templates. Initial PCR analysis, usi ng primers flanking the repeats, of DNA from two cloned stocks of the paras ite has shown that two DNA fragments of different size were amplified from each stock. Analysis of the inheritance of these fragments into the Fl prog eny of crosses demonstrated that the different size fragments were alleles that segregated in a Mendelian manner. The alleles at each of the three loc i segregated independently consistent with their localisation on three diff erent chromosomes. Analysis of a series of cloned isolates from tsetse dies showed that these loci were highly variable giving heterozygosities of 94% and the identification of 12 distinct alleles in a sample of 17 cloned iso lates. In order to determine whether isolates are heterogeneous in terms of trypanosome genotype, the allelic Variation at these three loci was examin ed in uncloned samples from tsetse flies isolated in Kiboko, Kenya and Luga la, Uganda. A significant proportion of the isolates (36% in Lugala and 47% in Kiboko) contained more than two alleles at one or more of the loci thus demonstrating that a high proportion of tsetse flies were infected with mo re than one genotype of trypanosomes. This was established, unequivocally, for two isolates by generating a series of cloned trypanosome lines from ea ch and determining the genotype of each clone; one isolate (927) contained seven different genotypes with a high proportion of the possible combinatio ns of alleles at each locus. These results indicate the possibility of freq uent genetic exchange in the field, they imply that a significant proportio n of mammalian hosts must contain mixtures of different trypanosome genotyp es and they demonstrate the advantages of using minisatellite markers for t he analysis of the population structure of T. brucei. (C) 1999 Elsevier Sci ence B.V. All rights reserved.