Molecular characterization of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense isolates and responses of Glossina pallidipes to Trypanosoma brucei brucei challenge

Abstract

African trypanosomiases are a group of related diseases that affect humans (Human African Trypanosomiasis - HAT) and their livestock (Animal African Trypanosomiasis), with devastating medical and economic consequences for Africa. The diseases are caused by trypanosome parasites cyclically transmitted by tsetse flies. Recent reports indicate that HAT is on the decline in sub Sahara Africa. However, the disease still remains a major health problem in some parts of Africa such as South Sudan, Democratic Republic of Congo and Uganda. Reports also indicate repeated irregular T. b. rhodesiense out breaks in traditionally endemic areas in south-east Uganda, with continued spread to previously unaffected areas in central Uganda. Although the disease has been reported to spread to new areas in Uganda, observations in the field indicate that the infection rates in tsetse flies are very low. Competence of the flies to transmit the parasite is determined by host midgut responses that either leads to clearance (self-cure) or establishment of the parasite infections. Investigations were conducted to determine evolutionary dynamics behind the origin of new foci and the impact of host species on parasite genetic diversity in Uganda. Trypanosoma brucei isolates {N = 269, n= 58(for Samples isolated from cattle)} were collected from different areas in Uganda and western Kenya and genotyped at 17 genetic marker loci (microsatellite). Analysis was carried out using Bayesian clustering and Discriminant Analysis of Principal Componens. Presence of serum resistance associated (SRA) gene in the isolates was determined using PCR. Results showed that the genotyped trypansosome isolates partitioned into three distinct genetic clusters. Clusters 1 and 3 included isolates from central and southern Uganda, whereas cluster 2 was composed of mainly isolates from western Kenya. FST values between sampling sites ranged from 0 to 0.67 while between the three genetic structures it ranged from0.24 to 0.46 with most values being statistically significant P<0.01. AMOVA results at p<0.05 indicated that the genetic variation was apportioned within (71.8%) rather than among the three clusters. These analyses revealed genetic admixture among the three genetic clusters and long-range dispersal, suggesting recent and possibly on-going gene flow between them and the new foci of HAT in central Uganda is as a result of northward movement of Trypanosoma brucei rhodesiense from the traditionally endemic foci. Therefore disease control efforts need to be enhanced to prevent continued spread to new foci. To determine molecular responses in Glossina pallidipes challenged with trypanosomes, teneral female Glossina pallidipes flies were challenged with Trypanosoma brucei brucei and vii dissected at 24 or 48 hours post challenge (hpc). Tissues were collected and analysed to establish key molecular responses mediating initial phase of establishment of the parasite in the fly. Transcriptomes of midguts and respective carcasses from the challenged and unchallenged flies were sequenced on illumina RNA-Seq platform, and analyzed for differentially expressed transcripts by mapping the RNA-Seq reads on G. pallidipes gene models. The transcripts were annotated and examined for enrichment of gene categories using, heat maps, BLAST2GO and R spider network software. Results from this study indicated that most of the differentially expressed transcripts at 24 hpc were associated with lipid remodeling/lipogenesis, proteolysis, urea cycle, carnitine trafficking, collagen metabolism, apoptosis, and cell growth/differentiation. Transcripts associated with 48 hpc included those linked to embryonic growth and development, muscle/motility, suppression of tumor, serine endopeptidase and related proteosomal degradation of target protein, enhanced translation of mRNA and neuronal development. There was pronounced expression of immune responsive transcripts 48 relative to 24 hpc, indicative of gradual maturity of immune responses in the fly or institution of vector-parasite endemic stability in the guts to facilitate the establishment of infection. Overall there was a systematic suppression of immunity in the G. pallidipes midgut in the initial phase of T. b. brucei challenge, which potentially facilitated initial establishment of the infection. Gradual and sequential immunological responses subsequently emerge contingent with the durations of challenge.