Abstract
BackgroundThe population structure and role of genetic exchange in African trypanosomes have been previously analyzed albeit with contradictory findings. To further investigate the role of genetic polymorphism on the population genetic structure of Trypanosoma b. rhodesiense, we hypothesized that parasite genotypes are clonal and stable over time.MethodsWe have undertaken a microsatellite marker analysis of T. b. rhodesiense isolates in a relatively new active HAT focus in Uganda (Kaberamaido-Dokolo-Amolatar) over a six-year period (2006–2012). We amplified six microsatellite markers by PCR directly from blood spotted FTA cards following whole genome amplification.ResultsThe majority of loci demonstrated an excess of heterozygosity (Ho > He, FIS < 0). We identified 26 unique genotypes among the 57 isolates, accounting for 45.6 % genotypic polymorphism. The presence of a high proportion of samples with repeated genotypes (54.4 %, 31/57), disagreement with Hardy-Weinberg equilibrium, and significant linkage disequilibrium between loci pairs, provide evidence that T. b. rhodesiense isolates from this focus are clonal. Our results show low values of FST’ (0–0.115) indicating negligible genetic differentiation across temporal isolates. Furthermore, predominant genotypes isolated in 2006 were still detectable in 2012.ConclusionsOur findings confirm the notion that endemicity is maintained by stable genotypes rather than an influx of new genotypes. Our results have considerable importance in understanding and tracking the spread of sleeping sickness with significant implication to disease control.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1542-1) contains supplementary material, which is available to authorized users.
Highlights
The population structure and role of genetic exchange in African trypanosomes have been previously analyzed albeit with contradictory findings
In order to determine if mating occurred among T. brucei isolates in east Africa, Hide et al [7] analyzed T. b. rhodesiense isolates from Uganda using multi-locus enzyme electrophoresis and reported an epidemic population structure
Microsatellite markers analysis We genotyped a total of sixty-three infected blood samples isolated from an active human African trypanosomiasis (HAT) focus in Northern Uganda over a six-year period (2006–2012) using six single-locus microsatellite markers
Summary
Ethical statement Ethical review of this retrospective study was by the Institutional Review Board of the Vector Control Division, Ministry of Health; final approval was provided by the Uganda National Council for Science and Technology. For WGA, 20 μl of the denatured cell lysate DNA was added to the Genomiphi V3 cake and samples incubated at 30 °C for 2 h followed by heating at 65 °C for 10 min with subsequent cooling at 4 °C. Polymerase chain reaction (PCR)-based genotyping Genotyping was done using six microsatellite loci previously shown to be polymorphic; Ch1/18, Ch2/5, Ch2/ PLC, Ch3/5 L5, Ch5/JS2 [10, 17, 19, 21] and M6C8 [24, 25]. Allele size determination and multi-locus genotype determination One primer of every second round pair for the nested PCR included a 5′-M13 or FAM modification in order to allow size separation of products utilizing a capillary based sequencer, the 3500xL Genetic Analyzer (Applied Biosystems). To evaluate temporal genetic differentiation we performed an analysis of molecular variance (AMOVA) and principal component analysis as implemented in GenAIEx v6.5 [26]
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