Are coinfections a threat to drug control programmes for livestock trypanosomes?

African trypanosomes cause substantial economic cost to livestock production in sub-Saharan Africa exacerbating poverty in afflicted regions. Three pathogenic species co-circulate, Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax, with infections being managed by chemoprophylaxis and drug therapy. However, coinfections between the species are common and several literature reports and our own data indicate that coinfection between trypanosome species and strains can ameliorate disease pathology with respect to monoinfections with a single species or strain. This generates a risk in settings where there is differential drug sensitivity among coinfecting trypanosomes exposed to suboptimal dosing, or where drug resistance is present in some circulating parasite populations. Specifically, by reverting coinfections to monoinfections comprising only the more resistant strain or species, drug intervention may lead to enhanced pathology. Consequently, drug application may generate a perverse outcome of increased disease.

In this proposal we will explore the interaction between coinfection, drug sensitivity and pathology in both a mouse model and disease-relevant livestock host. Specifically, we will:

1. Engineer drug resistant and sensitive Trypanosoma brucei and Trypanosoma congolense, respectively, using a known molecular resistance/sensitivity mechanism for diminazene, the most commonly used therapy for livestock trypanosomes. Specifically, the diminazene resistance determinant TbAT1, a nucleoside transporter, will be deleted in T. brucei to generate resistant parasites and T. congolense will be engineered to heterologously express the T. brucei AT1 gene, generating a diminazene super-sensitive line. This will allow the precision removal of T. congolense in experimental coinfections with T. brucei for our studies, avoiding the complexity of variable diminazene sensitivities different wild type laboratory and field strains may exhibit.

2. The engineered lines will be used to evaluate the impact of coinfection, or coinfection followed by diminazene induced monoinfections, on the proportion and distribution of the trypanosome populations and their pathology in mice. This will be achieved by quantitating parasite numbers for each species and reservoirs of T. brucei by IVIS imaging. Murine pathology will be scored by established criteria.

3. The engineered lines will be used to test how coinfection, or coinfection followed by diminazene-induced monoinfections, affects parasite prevalence and pathology in the disease-relevant bovine host using unique and dedicated large animal containment facilities at Roslin Institute.

The studies will determine the consequences for parasite prevalence and host pathology when a coinfection is redirected to a monoinfection through therapeutic intervention. This could be unexpectedly harmful for livestock health where differential resistance exists in mixed infection scenarios. Our experiments could prioritise epidemiological studies of this previously overlooked threat, and promote strategies to optimise dosing, or to treat diseased animals and sustain therapeutic efficacy. This would also accelerate the targeted adoption of alternative trypanocides as they become available.

The potential for adverse impact of therapeutic intervention in coinfection settings is unanticipated among farmers and policymakers in sub-Saharan Africa and may have been overlooked or dismissed as anecdote. We will ensure our findings are disseminated to the scientific community, policymakers and famers through our planned outreach activities and collaborations focused on livestock trypanosomes. These include planned meetings, for example a meeting Morrison is organising in Tanzania in 2023, and collaborative work with the Bill and Melinda Gates Foundation, links with the International livestock research Institute in Kenya and ongoing field work in Africa.

We aim to investigate coinfection, and reversion to monoinfection after drug therapy, for African trypanosomes and its impact on host pathology. To achieve this, we will (i) exploit the ability to genetically modify two trypanosome species, Trypanosoma brucei and Trypanosoma congolense and (ii) existing molecular knowledge of resistance mechanisms for the commonly used trypanocide diminazene. The nucleoside transporter AT1 is the major determinant of diminazene resistance in T. brucei such that its deletion confers resistance. Conversely, T. congolense can be engineered to be super-sensitive to diminazene through expression of T. brucei AT1. This enables robust selection of resistant and sensitive parasites in vivo in the presence or absence of diminazene and so the ability to precisely study either coinfections (without drug exposure) or monoinfections (after drug exposure of a coinfection with resistant and sensitive parasites).

Once engineered parasites are derived and validated, the prevalence of each species in a coinfection will be evaluated and the effective reversion to monoinfection confirmed after drug treatment. The parasite proportions, in vivo distribution of parasites and pathology will also be evaluated in a well-controlled murine model. Thereafter, to provide relevance to the natural host, parasite-induced pathology will be monitored in livestock with monoinfections, coinfections or with coinfections drug treated to restore monoinfections.

These analyses use existing methodologies routinely operational in our laboratory and exploit well-understood mechanisms of drug resistance for a commonly used trypanocide to assess the interaction of coinfection, drug resistance and drug therapy for the health of infected hosts, including livestock. The outcomes will inform how coinfection and drug resistance, or suboptimal dosing, may threaten the health benefits of therapeutic intervention for African trypanosomiases in livestock.