An integrated approach to tackling drug resistance in livestock trypanosomes.

Drug resistance is an increasing problem for many diseases worldwide. Trypanosomes are tsetse-fly transmitted single-celled organisms that cause serious disease in cattle - African Animal Trypanosomiasis (AAT), mainly in sub-Saharan Africa, where approximately 60 million cattle are at risk and AAT kills 3 million each year. The main measure farmers have to combat AAT is drug treatment, but we only have two main drugs, both of which are >50 years old and widely used. Resistance to these drugs is increasingly reported, and there are very few drugs in the development pipeline, meaning the current control methods have very doubtful sustainability. Despite being such a significant issue, we know very little about how trypanosomes resist the effect of the drug, and how AAT drug resistance emerges and spreads. Therefore we are limited in our ability to deploy informed strategies to mitigate the problem.

This project aims to generate data and resources to bridge these knowledge gaps. The applicants have been working in an area in Northern Tanzania since 2011. Recently farmers have been reporting drug treatment failure and the need to use increasing amounts of the prophylactic drug Isometamidium chloride (ISM) more frequently, suggesting emerging resistance.We already have samples from 5,000 cattle and 10,000 tsetse flies, as well as information on farmer drug use, from a cohort of farms in the area sampled between 2011 and 2017. Additionally, we have developed resources and capabilities for working on the relevant trypanosome species, Trypanosoma congolense, in the laboratory (most information derives from the human-infective Trypanosoma brucei, but it is increasingly apparent that these are very distinct organisms). This platform of preliminary data and resources will be used to answer our central hypothesis, that the management of animal trypanosomiasis in Tanzania is threatened by an emerging failure of ISM to provide adequate prophylaxis.

To test this hypothesis, we will address four main research questions: (i) what are the likely mechanisms by which resistance to ISM occurs and can we identify a marker?; (ii) what are the extent and cause of ISM failure?; (iii) what are the epidemiological consequences of ISM failure?; and (iv) how might resistance to trypanocides, including new drugs, be prevented? To achieve this we will: (1) identify ISM resistance mechanisms by generating resistant parasites in the laboratory and comparing resistant and susceptible parasites using biochemical, molecular and genomic analysis; (2) collect field data in the same area in order to assess drug use and drug quality, isolate drug resistant parasites, and assess the epidemiology of drug use and drug resistance in the field; (3) using data combined from the laboratory and the field, generate a mathematical model whose parameters are informed by both the laboratory and field data, allowing us to accurately assess how resistance emerges and spreads in AAT in the field; and (4) apply the model and predict scenarios that will inform on the selection and spread of resistance for a new trypanocidal compound in development by our industrial partners, GALVmed.

The outputs of this project would provide unprecedented and detailed insight into the epidemiology of AAT drug resistance, uncover mechanisms of drug resistance in the disease-relevant trypanosome species (including potential markers), and will develop the first application of mathematical modelling, importantly using accurate parameters, to provide insights into the dynamics of AAT drug resistance emergence and spread. As well as providing novel insights, the outputs have the potential to inform drug development and drug usage, by identifying strategies that will have the best chances of mitigating resistance, and therefore maximising the lifetime of both existing and novel drugs.

African Animal Trypanosomiasis (AAT), caused by tsetse-transmitted Trypanosoma congolense and T. vivax, is a major constraint on sub-Saharan African agriculture and food security. Control relies on the use of two main drugs, Isometamidium chloride (ISM - prophylactic) and Diminazene aceturate (DZ - therapeutic), both introduced over 50 years ago. ISM treatment failure is increasing across Africa, raising serious concerns about the sustainability of future AAT control. There is one therapeutic class currently in development, the benzoxaboroles (GALVmed/Boehringer Ingelheim; Galvmed are industrial partners on this proposal). Despite the reliance on these drugs in AAT, the understanding of drug failures, resistance mechanisms and epidemiology are poor, making it difficult to develop evidence-based mitigation strategies. This multidisciplinary project will use field, laboratory and modelling studies to test the overarching hypothesis that management of animal and human trypanosomiasis is threatened by an emerging failure of ISM to provide adequate treatment and prophylaxis. ISM resistance mechanisms and markers will be investigated through comparative biochemical, molecular and genomic analyses of resistant and susceptible Trypanosoma congolense, whilst relative fitness in hosts and vectors will be assessed. Field data will be collected to quantify trypanocide usage and effectiveness, assess resistance, isolate resistant T. congolense and measure epidemiological parameters. Resistance and spread will be investigated in silico with an AAT resistance model parameterised with field and experimental data on epidemiology and transmission of resistant and susceptible T. congolense. Finally, these findings will be extended to explore resistance in the benzoxaboroles. This is an opportunity for a step change in understanding AAT resistance, and will lead to development of strategies to maximise the useful lifetime of ISM, as well as new trypanocides such as the benzoxaboroles.

This project will fill important knowledge gaps that are currently limiting the development of sustainable control strategies for animal African trypanosomiasis (AAT). Elucidating the mechanisms of T. congolense resistance to isometamidium chloride (ISM), identifying a marker for diagnosis, and crucially better understanding the emergence and spread of resistance, as proposed in this study, are essential steps towards effective and sustainable control, including optimal use of novel drugs. We anticipate this project will enable and contribute to wider discussions on sustainable use of drugs in AAT control and help to drive this as a priority. Hence, the economic and societal impacts from this work include:

(1) Impact on disease control policies for sustainable use of trypanocides, leading to impacts on livestock farming in developing countries through reducing detrimental effects of resistance emergence.
Ultimate beneficiaries of the project are subsistence farmers in sub-Saharan Africa, who are finding that current AAT treatments are no longer working, threatening their livelihoods and food security. Approximately 50 million cattle, plus millions of other livestock, are at risk of AAT in tsetse-infected across an area of ~10M km2. AAT impacts include reduced milk yields, meat production, fertility, and draught power as well as mortality, and are estimated to cost billions (US$) to the region annually - estimated at $2.5 billion to Eastern Africa alone. The disease severely impacts sub-Saharan regions where livestock rearing is the main livelihood of small communities, including many countries on the DAC list of least developed countries. Tanzania has the third largest livestock population in Africa, and a high proportion of poor livestock keepers, with >4 million cattle threatened by trypanosomiasis.
Livestock keepers currently use 35-70 million doses of trypanocides annually. Two primary options exist for treatment of AAT: Isometamidium chloride and Diminazene aceturate . Both drugs are >50 years old and reported resistance to them is widespread. Outputs from this project will provide local (veterinary services), national (Ministry of Agriculture, Livestock and Fisheries), global (AU-PATTEC, FAO) and donor (GALVmed, DFID, BMGF) organisations with evidence to back up decision-making on sustainable use of drugs in AAT control. This project was co-constructed with local veterinary services and livestock keepers in Serengeti District, and national decision-makers in Tanzania, who have identified effective and sustainable trypanocide use as a particular concern. The same trypanocide drugs used for AAT are also used to reduce T. brucei circulation in cattle, which can be reservoirs for human African trypanosomiasis. Sustainable use of these drugs therefore has added benefits in prevention of human disease.

(2) Impact upon academic and industry AAT drug discovery and development programmes.
We know very little about how resistance emerges and spreads in livestock trypanosomes. This project will significantly advance this knowledge, both in terms of characterising mechanisms and rate of resistance emergence to ISM, and furthering our very scanty knowledge of the epidemiology of resistance in the field. The development of a mathematical model, that is developed and based on reliable data, will both inform on the dynamics of resistance and spread of ISM, and importantly be applicable to predicting resistance emergence and spread for novel trypanocides (such as the candidate compound currently under development by project partners GALVmed) - this output is a critical gap in knowledge and capability at present, which would be able to inform strategies to minimise resistance emergence and spread, and maximise the lifetime of both ISM and novel trypanocides. The applicants have links with relevant academic, industrial and policy stakeholders to enable dissemination and uptake of results in order to translate impact to farmers.