The genetic diversity of trypanosoma cruzi, multiclonality of natural populations and the characterisation of Paraguayan isolates and experimentally derived T. cruzi I. hydrids
The aims of this PhD programme were: optimisation of conditions for clonal isolation of Trypanosoma cruzi from individual triatomine bugs and molecular characterisation of clonal isolates from field sites in Paraguay, with special consideration given to host/vector associations and transmission cycles, and further characterisation of putative parents and experimental hybrid progeny of T cruzi 1. with comparison of hybrid genotypes and field isolates from Paraguay. The development of an optimised solid medium plating technique allowed the resolution of mixed infections by the isolation of clonal colonies of T cruzi from single triatomine bugs and experimentally infected mice. Some nutrient plate types produced rapid growth with high plating efficiencies and longer colony viability in comparison with previous methods. Using this technique growth was also possible without the use of a CO2 incubator. Paraguayan isolates were characterised from mammal hosts, sylvatic T infestans, and domestic T infestans from several geographical locations. Isolates were characterised by phenotyping and by genotyping large and small subunit rRNA and mini-exon genes. Analysis enabled assignment of isolates into one of two major lineages (TCI or TC2), and further attribution to one of five subgroups of TC2 (a-e). In Paraguay isolates were all subgroups of TC2. We identified TC2b, TC2c. TC2d and Tc2e circulating in Paraguay. TC2b, TC2d and TC2e were present in domestic cycles (with a predominance of TC2e, 55.2%) and TC2c, TC2b and TC2d in sylvatic cycles. Two species of armadillo (Dasypus novemcinctus; TC2c, TC2d and Euphractus sexcinctus; TC2b, TC2d) were identified as sylvatic reservoirs of T cruzi. These findings accord with our theory that terrestrial armadillos and rodents may have an evolutionary association with TC2. Sylvatic colonies of the vector T infestans were identified from two locations in the Paraguayan Chaco. T cruzi infection in sylvatic T infestans was identified as TC2b. This vector was previously regarded as almost exclusively domestic. and this finding has significant implications for future vector surveillance programmes in Paraguay. By further characterisation of hybrid clones it has been proved for the first time that T cruzi has an extant capacity for genetic exchange. Two genetically transformed putative parental types and six double drug resistant clones, previously isolated by coinfection of mammal cells, were analysed by isoenzyme phenotype, random amplified polymorphic DNA (RAPDs), karyotype analysis and DNA sequencing. These results, together with parallel work performed within our group, suggest an unusual mechanism of genetic exchange involving fusion of parental genotypes and subsequent loss of alleles in polyploid progeny, in conjunction with homologous recombination, and uniparental inheritance of kinetoplast maxicircle DNA. There were parallels between experimentally generated hybrids and natural isolates. The implications of genome fusion and aneuploidy are profound and the mechanism is likely to be widespread providing a possible means of rapid speciation and evolution.