High Prevalence of Drug Resistance in Animal Trypanosomes without a History of Drug Exposure

Trypanosomosis is responsible for the death of 3 million heads of cattle yearly, with 50 million animals at risk in sub-Saharan Africa. DA, a commonly used drug against the disease, was marketed decades ago. Drug resistance is reported in 21 African countries. A common argument about the origin of drug resistance is the selection by the drug of rare individuals that are naturally resistant and the propagation of those individuals in the population because of the competitive advantage they have when exposed to drug. When the drug pressure decreases, the wild-type individuals regain their supremacy. The principal objective of this study was thus to estimate the prevalence of trypanosomes resistant to DA in a population that was never exposed to the drug. Our results showing a high prevalence of drug resistance in environments free of any drug pressure is thought provoking and suggests that ceasing the use of DA will not allow for a return to a DA-sensitive population of trypanosomes. Drug resistance in animal trypanosomes thus present a pattern different from what is observed with Plasmodium sp. (causative agent of malaria) where a complete stoppage in the use of the chloroquine allows for a return to drug sensitivity

Improved PCR-RFLP for the Detection of Diminazene Resistance in Trypanosoma congolense under Field Conditions Using Filter Papers for Sample Storage

Animal African trypanosomiasis (AAT) is caused by different species of the pro- tozoan parasite Trypanosoma and affects a wide range of domestic animals. Trypano- soma congolense is widespread in the whole of sub-Saharan Africa and is the species causing considerable losses in livestock pro- duction, often affecting the health status of humans through endangering the food supply of rural communities. It is estimated that 50 million cattle are at risk of the disease and that the direct and indirect annual losses related to AAT reach US$4.5 billion [1]

Réseau d'EpidémioSurveillance de la Chimiorésistance aux trypanocides et aux acaricides en Afrique de l'Ouest (RESCAO)

Epidemiological Monitoring Network of Chemoresistance to Trypanocidal and Acaricides Drugs in West Africa (RESCAO). To better coordinate the efforts against trypanocidal and acaricides drugs resistance, the Institute of Tropical Medicine (ITM) of Antwerp and the "Centre International de Recherche-Développement sur l'Elevage en zone Subhumide (CIRDES)" of Bobo Dioulasso, established in April 2009 an epidemiological surveillance network of chemoresistance to trypanocidal and acaricides drugs in Western Africa, named RESCAO. Its main objective is to contribute to the improvement of the livestock health and of the productivity of agriculture in tropical Africa, through both an efficient strategic control of trypanosomosis and tick born diseases, including a rational use of the available therapeutic drugs. RESCAO is headed by a regional steering committee based at CIRDES. This committee meets on a yearly basis to overview the on-going activities and to identify new strategies for action. Moreover, molecular analyzes performed on samples from seven West African's countries, members of RESCAO, have shown that resistance to diminazeneaceturate was widespread in Trypanosoma congolense with percentages ranging from 67.85 (19/28) for Burkina Faso to 100% (9/9) for Ghana

Assessment of animal African trypanosomiasis (AAT) vulnerability in cattle-owning communities of sub-Saharan Africa

Background: Animal African trypanosomiasis (AAT) is one of the biggest constraints to livestock production and a threat to food security in sub-Saharan Africa. In order to optimise the allocation of resources for AAT control, decision makers need to target geographic areas where control programmes are most likely to be successful and sustainable and select control methods that will maximise the benefits obtained from resources invested. Methods: The overall approach to classifying cattle-owning communities in terms of AAT vulnerability was based on the selection of key variables collected through field surveys in five sub-Saharan Africa countries followed by a formal Multiple Correspondence Analysis (MCA) to identify factors explaining the variations between areas. To categorise the communities in terms of AAT vulnerability profiles, Hierarchical Cluster Analysis (HCA) was performed. Results: Three clusters of community vulnerability profiles were identified based on farmers’ beliefs with respect to trypanosomiasis control within the five countries studied. Cluster 1 communities, mainly identified in Cameroon, reported constant AAT burden, had large trypanosensitive (average herd size = 57) communal grazing cattle herds. Livestock (cattle and small ruminants) were reportedly the primary source of income in the majority of these cattle-owning households (87.0 %). Cluster 2 communities identified mainly in Burkina Faso and Zambia, with some Ethiopian communities had moderate herd sizes (average = 16) and some trypanotolerant breeds (31.7 %) practicing communal grazing. In these communities there were some concerns regarding the development of trypanocide resistance. Crops were the primary income source while communities in this cluster incurred some financial losses due to diminished draft power. The third cluster contained mainly Ugandan and Ethiopian communities which were mixed farmers with smaller herd sizes (average = 8). The costs spent diagnosing and treating AAT were moderate here. Conclusions: Understanding how cattle-owners are affected by AAT and their efforts to manage the disease is critical to the design of suitable locally-adapted control programmes. It is expected that the results could inform priority setting and the development of tailored recommendations for AAT control strategies

A major genetic locus in <i>Trypanosoma brucei</i> is a determinant of host pathology

The progression and variation of pathology during infections can be due to components from both host or pathogen, and/or the interaction between them. The influence of host genetic variation on disease pathology during infections with trypanosomes has been well studied in recent years, but the role of parasite genetic variation has not been extensively studied. We have shown that there is parasite strain-specific variation in the level of splenomegaly and hepatomegaly in infected mice and used a forward genetic approach to identify the parasite loci that determine this variation. This approach allowed us to dissect and identify the parasite loci that determine the complex phenotypes induced by infection. Using the available trypanosome genetic map, a major quantitative trait locus (QTL) was identified on T. brucei chromosome 3 (LOD = 7.2) that accounted for approximately two thirds of the variance observed in each of two correlated phenotypes, splenomegaly and hepatomegaly, in the infected mice (named &lt;i&gt;TbOrg1&lt;/i&gt;). In addition, a second locus was identified that contributed to splenomegaly, hepatomegaly and reticulocytosis (&lt;i&gt;TbOrg2&lt;/i&gt;). This is the first use of quantitative trait locus mapping in a diploid protozoan and shows that there are trypanosome genes that directly contribute to the progression of pathology during infections and, therefore, that parasite genetic variation can be a critical factor in disease outcome. The identification of parasite loci is a first step towards identifying the genes that are responsible for these important traits and shows the power of genetic analysis as a tool for dissecting complex quantitative phenotypic traits

In vitro activity of salinomycin and monensin derivatives against Trypanosoma brucei

Background: African trypanosomes are the causative agents of sleeping sickness in humans and nagana disease in livestock animals. As the few drugs available for treatment of the diseases have limited efficacy and produce adverse reactions, new and better tolerated therapies are required. Polyether ionophores have been shown to display anti-cancer, anti-microbial and anti-parasitic activity. In this study, derivatives of the polyether ionophores, salinomycin and monensin were tested for their in vitro activity against bloodstream forms of Trypanosoma brucei and human HL-60 cells. Results: Most polyether ionophore derivatives were less trypanocidal than their corresponding parent compounds. However, two salinomycin derivatives (salinomycin n-butyl amide and salinomycin 2,2,2-trifluoroethyl ester) were identified that showed increased anti-trypanosomal activity with 50% growth inhibition values in the mid nanomolar range and minimum inhibitory concentrations of below 1 μM similar to suramin, a drug used in the treatment of sleeping sickness. In contrast, human HL-60 cells were considerably less sensitive towards all polyether ionophore derivatives. The cytotoxic to trypanocidal activity ratio (selectivity) of the two promising compounds was greater than 250. Conclusions: The data indicate that polyether ionophore derivatives are interesting lead compounds for rational anti-trypanosomal drug development

Reduced Mitochondrial Membrane Potential is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo- ATPase

Background: Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine. Methodology/Principal Findings: Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance. Conclusions/Significance: Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential

Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug clas

Irradiated Male Tsetse from a 40-Year-Old Colony Are Still Competitive in a Riparian Forest in Burkina Faso

Background Tsetse flies are the cyclical vectors of African trypanosomosis that constitute a major constraint to development in Africa. Their control is an important component of the integrated management of these diseases, and among the techniques available, the sterile insect technique (SIT) is the sole that is efficient at low densities. The government of Burkina Faso has embarked on a tsetse eradication programme in the framework of the PATTEC, where SIT is an important component. The project plans to use flies from a Glossina palpalis gambiensis colony that has been maintained for about 40 years at the Centre International de Recherche-Développement sur l'Elevage en zone Subhumide (CIRDES). It was thus necessary to test the competitiveness of the sterile males originating from this colony. Methodology/Principal Findings During the period January-February 2010, 16,000 sterile male G. p. gambiensis were released along a tributary of the Mouhoun river. The study revealed that with a mean sterile to wild male ratio of 1.16 (s.d. 0.38), the abortion rate of the wild female flies was significantly higher than before (p = 0.026) and after (p = 0.019) the release period. The estimated competitiveness of the sterile males (Fried index) was 0.07 (s.d. 0.02), indicating that a sterile to wild male ratio of 14.4 would be necessary to obtain nearly complete induced sterility in the female population. The aggregation patterns of sterile and wild male flies were similar. The survival rate of the released sterile male flies was similar to that observed in 1983-1985 for the same colony. Conclusions/Significance We conclude that gamma sterilised male G. p. gambiensis derived from the CIRDES colony have a competitiveness that is comparable to their competitiveness obtained 35 years ago and can still be used for an area-wide integrated pest management campaign with a sterile insect component in Burkina Faso. (Résumé d'auteur