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Graphical abstractHighlights► Diminazene transporter in Trypanosoma congolense has been proposed to be TcoAT1. ► Here, TcoAT1 was cloned and functionally expressed in Trypanosoma brucei. ► TcoAT1 did not mediate the uptake of diminazene, only of purine nucleosides. ► Expression of TcoAT1 did not alter drug sensitivity in trypanosomes. ► We conclude that TcoAT1 is a transporter for purine nucleosides, not for diminazene.

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There are currently 17 African countries in which animal trypanocidal drug resistance has been reported. Largescale surveys were carried out in only ten of them. The lack of baseline information is mainly due to the fact that the methods currently available for the detection of drug resistance are laborious, expensive and time consuming. In this review the mechanisms involved in resistance to isometamidium and diminazene will be discussed, together with some new molecular detection tools that have been developed recently enabling faster diagnosis of drug resistance than conventional laboratory or field tests. African animal trypanosomiasis and drug resistanceTsetse fly-transmitted trypanosomiasis is an important constraint to livestock development in sub-Saharan Africa with estimated annual losses owing to the direct and indirect effects of the disease running into billions of dollars. Approximately 9 million km 2 of sub-Saharan Africa, representing about one-third of the total land, is affected by tsetse flies [1]. Within this region, some 46-62 million head of cattle and other livestock species are at risk of the disease [2]. Trypanosomiasis is controlled either by controlling the vector or by controlling the parasite, or a combination of both. Over the years, a large arsenal of vector-control tools has been developed. Nevertheless, the control of animal trypanosomiasis in often poor rural communities has and will continue to rely heavily on the use of trypanocidal drugs. This is not surprising considering the private nature (i.e. the easy, individual, nonconcerted use) of such treatments and the difficulties in maintaining cleared areas in the absence of barriers to re-invasion of tsetse flies. Only a small group of chemoprophylactic and chemotherapeutic trypanocidal compounds are currently in use and new compounds are unlikely to become available in the near future [3]. Geerts and Holmes [4] estimated that in Africa 35 million doses of veterinary trypanocidal drugs are administered each year with isometamidium chloride (ISM), ethidium bromide (EtBr) and diminazene aceturate (DA) estimated to represent 40%, 26% and 33%, respectively, of the total trypanocidal drug market by value [5]. ISM is mainly used as a prophylactic drug and provides on average 3 months' protection (2-22 weeks) against trypanosome infection. DA has only therapeutic properties and EtBr has limited prophylactic properties and is mainly used as a therapeutic agent [6]. Considering the well known mutagenic properties of EtBr, this drug should ideally be removed from the drug market, but in practice it is still widely used in many countries. Removing this drug from the market would not jeopardize the treatment of animal trypanosomiasis because it can be replaced either by DA for curative purposes or by ISM for prophylactic purposes. When trypanosomes are resistant to ISM, EtBr will be ineffective as cross-resistance is observed between the two drugs [7]. As a result of inappropriate trypanocidal drug-use practices, there is growing co...

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BackgroundTrypanosomosis caused by Trypanosoma congolense is a major constraint to animal health in sub-Saharan Africa. Unfortunately, the treatment of the disease is impaired by the spread of drug resistance. Resistance to diminazene aceturate (DA) in T. congolense is linked to a mutation modifying the functioning of a P2-type purine-transporter responsible for the uptake of the drug. Our objective was to verify if the mutation was linked or not to drug pressure.Methodology/Principal FindingsThirty-four T. congolense isolates sampled from tsetse or wildlife were screened for the DA-resistance linked mutation using DpnII-PCR-RFLP. The results showed 1 sensitive, 12 resistant and 21 mixed DpnII-PCR-RFLP profiles. This suggests that the mutation is present on at least one allele of each of the 33 isolates. For twelve of the isolates, a standard screening method in mice was used by (i) microscopic examination, (ii) trypanosome-specific 18S-PCR after 2 months of observation and (iii) weekly trypanosome-specific 18S-PCR for 8 weeks. The results showed that all mice remained microscopically trypanosome-positive after treatment with 5 mg/kg DA. With 10 and 20 mg/kg, 8.3% (n = 72) and 0% (n = 72) of the mice became parasitologically positive after treatment. However, in these latter groups the trypanosome-specific 18S-PCR indicated a higher degree of trypanosome-positivity, i.e., with a unique test, 51.4% (n = 72) and 38.9% (n = 72) and with the weekly tests 79.2% (n = 24) and 66.7% (n = 24) for 10 and 20 mg/kg respectively.Conclusion/SignificanceThe widespread presence of the DA-resistance linked mutation in T. congolense isolated from wildlife suggests that this mutation is favourable to parasite survival and/or its dissemination in the host population independent from the presence of drug. After treatment with DA, those T. congolense isolates cause persisting low parasitaemias even after complete elimination of the drug and with little impact on the host's health.

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FFLB identified the trypanosomes in 65 of 105 (61.9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main clade of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

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