A novel metabarcoded deep amplicon sequencing tool for the detection of diminazene drug resistance mutations in Trypanosoma evansi

Diminazene resistance of Trypanosoma is an increasing problem whose underlying genetics are not fully understood. The development of diminazene resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 aminopurine due to alteration in the corresponding adenosine transporter-1 (AT-1) domain. Specific mutant-type alleles (71V, 178T, 181E, 239G, 286S and 380P) linked to resistance were more prevalent in T. brucei subspecies gambiense in humans. In the present study, we have analysed the adenosine transporter-1 (AT-1) domain of the P2 aminopurine from Trypanosoma evansi to explore a possible genetic link between the presence of mutations in this gene and diminazene treatment in ruminants. The genetic basis of diminazene resistance was investigated with a new high-throughput metabarcoded deep amplicon sequencing tool in T. evansi field isolates. We examined Trypanosoma isolates collected from ruminants (Camel, Buffalo, Cattle, Goat, Sheep) in seven known endemic regions of the Punjab province of Pakistan, where animals are treated in a sporadic manner, often with generic diminazene drugs of unknown quality. Blood samples were collected for DNA preparation, adapter/barcoded PCR amplification and Illumina Mi-Seq based sequencing. Our results indicate that 7 T. evansi field isolates had resistance mutations at codon 178E/S, 239Y/A/E, 286S/H/I/D/T in the adenosine transporter-1 (AT-1) domain. In contrast, 26 T. evansi field isolates had susceptible mutations at codon A178, G181, D239 and N286 in the adenosine transporter-1 (AT-1) domain. The overall outcomes will be useful in understanding the emergence of diminazene resistance. This information will help design strategies to optimize the use and lifespan of the most important drug to control trypanosomiasis.