Detecting drug resistance in surra in India.

Surra is a disease of multiple different mammals, including key livestock animals such as horses, pigs, sheep, goats, buffalo and cattle. It is caused by parasitic trypanosomes of the Trypanosoma evansi species, that should be, according to some specialists, classified as a sub-species of Trypanosoma brucei given its evolutionary derivation from this latter parasite. T. evansi has undergone key physiological alterations when compared to T. brucei brucei, including loss of maxi-circle DNA, corresponding to the key coding capability of the parasites' kinetoplast (mitochondrial) DNA. Since the mitochondrion's role is largely suppressed in parasites that dwell in the mammalian bloodstream, but critical for viability in the tsetse fly that is responsible for cyclical transmission of T. brucei brucei, T. evansi has lost this cyclical transmission. This loss appears to have been accompanied by ill-defined gain of ability to infect a wide range of mammalian species and also to be mechanically transmitted through other biting arthropods. This has enabled T. evansi, and the disease it causes, to spread globally, being endemic across much of north Africa, Asia, and Latin America, with sporadic cases also occurring in parts of Europe. The full economic consequences of surra are not well understood either. Estimates of approaching $700m U.S in lost animal production in India are probably an under-estimate because, in many instances, infections are mild, particularly in cattle and, as such, may go undetected, and yet have significant effects on growth and fecundity of affected animals. Given the prevalence of the parasites across much of Asia and Latin America the global economic losses likely run into several billions of dollars each year.
The drugs used against surra (including diminazene aceturate, isometamidium and quinapyramine) have been available for many years. They are all trypanocides that have been used against other veterinary trypanosomiases including the animal African trypanosomiases caused by Trypanosoma brucei, T. congolense and T. vivax. Systematic analysis of their suitability in surra has never been conducted and, indeed, given the role of the mitochondrion and kinetoplast DNA in the activity of all of these drugs, questions about their suitability have been raised. The length of time in use and poorly regulated regimens have, inevitably, raised antimicrobial resistance to these trypanocidal drugs. Again, however, a dearth of studies makes it difficult to offer clear evidence on the incidence and distribution of T. evansi resistance to these drugs.
Here we propose to systematically determine the molecular mechanisms of resistance to these trypanocidal drugs and identify genetic changes associated with resistance. We will also determine whether a new class of trypanocidal compounds, the valinate amide benzoxaboroles, that have potent efficacy against trypanosomes (including T. brucei, T. congolense and T. vivax) are also efficacious against T. evansi. Moreover, we will ascertain whether the mode of action (inhibition of the RNA processing enzyme CPSF3, is conserved in T. evanis, and also whether resistance comes about the same way is it does in other trypanosomes (through loss of an enzyme that processes the parent compound within the cell.
If this mechanism exists we propose to develop molecular probes that can detect the activity of the processing enzyme and thus determine resistance to these compounds. Moreover, for diminazene aceturate, we also believe that existing fluorescent tests can report on resistance too. For the other drugs we hope to develop tests when we have determined resistance mechanisms.
Our work will also extend to conduct a survey of the epidemiological incidence of surra in livestock in selected parts of India and also, with genetic and fluorescent tests to ascertain the presence and distribution of resistance to existing drugs.

Surra is a disease, present in numerous mammalian species, caused by the protozoan parasite Trypanosoma evansi. Drugs against surra have been used for many years and treatment failures are relatively common. However, no systematic analysis on the causes of antimicrobial resistance in surra have been conducted and the prevalence of the problem is unknown. We propose to identify modes of action and resistance mechanisms to three drugs currently used to treat surra. We will also confirm the potency of a new class of benzoxaborole prodrugs that offer a new route to treatment, and identify mode of action and resistance mechanisms for these compounds too. For diminazene aceturate, we have shown in the related parasite T. equiperdum, that mutations to the TeqAT1 gene that encodes an aminopurine transporter responsible for diminazene uptake is lost in resistance, as in T. brucei. T. evansi has the same gene, hence loss of uptake due to TeAT1 mutation is the likely the cause of resistance. We will confirm this by selecting several diminazene resistant T. evansi lines and sequencing these and existing ones. We have a fluorescence test that rapidly shows loss of uptake via the transporter and thus detects resistance. For the benzoxaboroles, analysis of the T. evansi genome indicates that the same CPSF3 target of the drug exists and the same CBP serine carboxypeptidase prodrug activator is present too. We will select de novo resistant lines to confirm this mechanism in T. evansi. We will then develop fluorescent probes (with a fluorophore and quencher separated by the CBP cleaved region of the valinate amide class) to create resistance tests for these compounds. We will also select isometamidium and quinapyramine resistance and sequence genomes of resistant lines to ascertain resistance mechanisms. Based on this we aim to develop new tests for resistance to these compounds. T. evansi isolates from India will be probed for resistance with the tests