Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor

Abstract

Background: The apicomplexan hemoparasite Theileria equi is a causative agent of equine piroplasmosis, eradicated from the United States in 1988. However, recent outbreaks have sparked renewed interest in treatment options for infected horses. Imidocarb dipropionate is the current drug of choice, however variation in clinical response to therapy has been observed.

Methods: We quantified the in vitro susceptibility of two T. equi isolates and a lab generated variant to both imidocarb dipropionate and a bumped kinase inhibitor compound 1294. We also evaluated the capacity of in vitro imidocarb dipropionate exposure to decrease susceptibility to that drug. The efficacy of imidocarb dipropionate for clearing infection in four T. equi infected ponies was also assessed.

Results: We observed an almost four-fold difference in imidocarb dipropionate susceptibility between two distinct isolates of T. equi. Four ponies infected with the less susceptible USDA Florida strain failed to clear the parasite despite two rounds of treatment. Importantly, a further 15-fold decrease in susceptibility was produced in this strain by continuous in vitro imidocarb dipropionate exposure. Despite a demonstrated difference in imidocarb dipropionate susceptibility, there was no difference in the susceptibility of two T. equi isolates to bumped kinase inhibitor 1294.

Conclusions: The observed variation in imidocarb dipropionate susceptibility, further reduction in susceptibility caused by drug exposure in vitro, and failure to clear T. equi infection in vivo, raises concern for the emergence of drug resistance in clinical cases undergoing treatment. Bumped kinase inhibitors may be effective as alternative drugs for the treatment of resistant T. equi parasites.

Figure 1

Nested PCR detection of T. equi infection in ponies after failed IMD treatment. (a) 2% agarose gel showing results from packed erythrocytes collected 16 days after the second round of IMD treatment. Lane 2: negative control erythrocytes from an uninfected horse (−). Lanes 3, 4, 5, 6: erythrocytes from the four IMD treated ponies. Lane 7: positive control erythrocytes from a known infected horse showing the 200 bp T. equi ema-1 amplicon (+). (b) T. equi ema-1 amplicons from packed erythrocytes collected 80 days after the second round of IMD treatment. Lanes 2, 3, 4, 5: erythrocytes from the four IMD treated ponies. Lane 6: negative control (−). Lane 7: positive control (+).

Figure 2

Flow cytometric scatter plots depicting percent parasitized erythrocytes (PPE). Representative samples from a 72 hour growth inhibition assay. Parasitized erythrocytes stained with hydroethidine were detected in the FL-2 channel, and are represented in the bottom right quadrant. (a) Normal uninfected erythrocytes stained with hydroethidine (negative control). (b) T. equi-infected erythrocytes incubated without imidocarb dipropionate (IMD; positive control). (c) T. equi-infected erythrocytes incubated with 2.76 μM IMD.

Figure 3

Variable in vitro susceptibility to IMD among different T. equi isolates. Each point on the nonlinear regression curve represents the mean percent of the maximum PPE determined in triplicate wells. IC₅₀ is drug concentration resulting in 50% of the maximum PPE detected in wells not containing drug. (a) Susceptibility of the USDA FL strain (red) and the TX isolate (purple). (b) Susceptibility of FL Exp variant 1 (purple) and FL Exp variant 2 (green).

Figure 4

Amino acid alignment through the ATP-binding domains of calcium dependent protein kinases. Includes amino acid sequences from P. falciparum (Pf), T. equi (Te), B. bovis (Bb), and T. gondii (Tg). The ATP-binding region is boxed, and the gatekeeper residues are shaded.

Figure 5

In vitro susceptibility of T. equi isolates to BKI compound 1294. Each point on the nonlinear regression curve represents the mean percent of the maximum PPE determined in triplicate wells. IC₅₀ is drug concentration resulting in 50% of the maximum PPE detected in wells not containing drug. Depicted results represent TX (purple) and FL Exp variant 1 (red).