A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR.

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.

Assessing the effect of insecticide-treated cattle on tsetse abundance and trypanosome transmission at the wildlife-livestock interface in Serengeti, Tanzania.

In the absence of national control programmes against Rhodesian human African trypanosomiasis, farmer-led treatment of cattle with pyrethroid-based insecticides may be an effective strategy for foci at the edges of wildlife areas, but there is limited evidence to support this. We combined data on insecticide use by farmers, tsetse abundance and trypanosome prevalence, with mathematical models, to quantify the likely impact of insecticide-treated cattle. Sixteen percent of farmers reported treating cattle with a pyrethroid, and chemical analysis indicated 18% of individual cattle had been treated, in the previous week. Treatment of cattle was estimated to increase daily mortality of tsetse by 5-14%. Trypanosome prevalence in tsetse, predominantly from wildlife areas, was 1.25% for T. brucei s.l. and 0.03% for T. b. rhodesiense. For 750 cattle sampled from 48 herds, 2.3% were PCR positive for T. brucei s.l. and none for T. b. rhodesiense. Using mathematical models, we estimated there was 8-29% increase in mortality of tsetse in farming areas and this increase can explain the relatively low prevalence of T. brucei s.l. in cattle. Farmer-led treatment of cattle with pyrethroids is likely, in part, to be limiting the spill-over of human-infective trypanosomes from wildlife areas.

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.

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.

The industrial melanism mutation in British peppered moths is a transposable element.

Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of 'jumping genes' as a source of major phenotypic novelty.

Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring.

BACKGROUND: As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys. METHODS AND FINDINGS: The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting. CONCLUSION: We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites.