Evidence of the absence of human African trypanosomiasis in two northern districts of Uganda: Analyses of cattle, pigs and tsetse flies for the presence of Trypanosoma brucei gambiense

Abstract

Background: Large-scale control of sleeping sickness has led to a decline in the number of cases of Gambian human African trypanosomiasis (g-HAT) to <2000/year. However, achieving complete and lasting interruption of transmission may be difficult because animals may act as reservoir hosts for T. b. gambiense. Our study aims to update our understanding of T. b. gambiense in local vectors and domestic animals of N.W. Uganda.

Methods: We collected blood from 2896 cattle and 400 pigs and In addition, 6664 tsetse underwent microscopical examination for the presence of trypanosomes. Trypanosoma species were identified in tsetse from a subsample of 2184 using PCR. Primers specific for T. brucei s.l. and for T. brucei sub-species were used to screen cattle, pig and tsetse samples.

Results: In total, 39/2,088 (1.9%; 95% CI = 1.9-2.5) cattle, 25/400 (6.3%; 95% CI = 4.1-9.1) pigs and 40/2,184 (1.8%; 95% CI = 1.3-2.5) tsetse, were positive for T. brucei s.l.. Of these samples 24 cattle (61.5%), 15 pig (60%) and 25 tsetse (62.5%) samples had sufficient DNA to be screened using the T. brucei sub-species PCR. Further analysis found no cattle or pigs positive for T. b. gambiense, however, 17/40 of the tsetse samples produced a band suggestive of T. b. gambiense. When three of these 17 PCR products were sequenced the sequences were markedly different to T. b. gambiense, indicating that these flies were not infected with T. b. gambiense.

Conclusion: The lack of T. b. gambiense positives in cattle, pigs and tsetse accords with the low prevalence of g-HAT in the human population. We found no evidence that livestock are acting as reservoir hosts. However, this study highlights the limitations of current methods of detecting and identifying T. b. gambiense which relies on a single copy-gene to discriminate between the different sub-species of T. brucei s.l.

Fig 1. Map of sampling sites for tsetse, cattle and pigs from N.W. Uganda.

Intervention zone denotes the area that was under tsetse control during the collection of samples described in this paper [27]. The map was created by authors for this publication using Gnu Image Manipulation Software [31].

Fig 2. Diagrammatic representation of the location of the multiplex ITS primers in relation to the universal primers designed by Adams et. al. (2006) on the ribosomal DNA.

Fig 3. Image showing the relative sizes of the mITS PCR reaction for T. congolense Savannah (1), T. vivax (2) and T. b. brucei (3), extraction negative control (4), reagent negative control (5) and a second T. b. brucei positive control (6).

Fig 4. Example of gel run showing the results of the sub-species Trypanozoon multiplex PCR.

The 324bp PLC product can be seen in the three positive controls and in sample number 2. The 669bp SRA product can be seen in the T. b. rhodesiense positive control but is absent from the other T. brucei sub-species. A >1 kb VSG product is visible in sample 2 and is faintly visible in the T. b. rhodesiense positive control. A fourth product of ~700 bp can also be seen in the T. b. gambiense and T. b. rhodesiense positive controls as well as sample 2.

Fig 5. Results of the sequence alignment of the three samples sent for sequencing (3923, 3861 and 4280) against the T. b. gambiense positive control (Pos_ctrl) and reference sequence (FN555990) from the NCBI database.

Image generated using MultAlin [38].