Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi

Abstract

Background: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective against Babesia microti, without showing toxicity in mammals.

Methods: The inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of the ELQ compounds on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay.

Results: We calculated the half maximal inhibitory concentration (IC₅₀) at 72 h, which ranged from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 among all cultured parasites tested at 72 h. None of the parasites tested were able to replicate in cultures in the presence of ELQ-300 and ELQ-316 at the maximal inhibitory concentration (IC₁₀₀), which ranged from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC₁₀₀ in in vitro testing.

Conclusions: The compounds ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

Keywords: Babesia bigemina; Babesia bovis; Babesia caballi; Bovine babesiosis; ELQ-300; Endochin-like quinolones; Equine piroplamsosis; Theileria equi; nnELQ-316.

Fig. 1

Parasite culture growth at 24 h (purple bars), 48 h (blue bars) and 72 h (yellow bars) without and after addition of different concentrations of endochin-like quinolone-300 (ELQ-300). a Babesia bovis, b B. bigemina c B. caballi, d Theilera equi. “0” represents parasites in the absence of ELQ-300. Assays were carried out in triplicate, and the error bars indicate standard error deviation for each ELQ-300 concentration tested. Asterisk (*) represents statistically significant difference between cultures grown without and with ELQ-300 at P < 0.05, using Student’s t-test. nRBC Non-infected erythrocytes, PPE percentage of parasitized erythrocytes

Fig. 2

Parasite culture growth at 24 h (purple bars), 48 h (blue bars) and 72 h (yellow bars) without and after addition of different concentrations of ELQ-316. a B. bovis, b B. bigemina, c B. caballi, d T. equi. “0” represents parasites in the absence of ELQ-316. Assays were carried out in triplicate, and the error bars indicate standard error deviation for each ELQ-316 concentration tested. Asterisk (*) represents statistically significant difference between cultures grown without and with ELQ-316 at P < 0.05, using Student’s t-test

Fig. 3

Parasite culture growth at 24 h (purple bars), 48 h (blue bars), and 72 h (yellow bars) using IC₁₀₀ of ELQ-300, and at 8 days (green bars). a B. bovis, b B. bigemina, c B. caballi, d T. equi. Upper panel of each figure part represents 0.2% PPE, lower panel 2% PPE. “0” represents parasites grown without addition of ELQ-300. Assays were carried out in triplicate and the error bars indicate standard error deviation. Asterisk (*) represents statistically significant difference between cultures grown without and with ELQ-300 at P < 0.05, using Student’s t-test. C₅₀ and IC₁₀₀ Half maximal and maximal inhibitory concentrations, respectively, DMSO dimethyl sulfoxide

Fig. 4

Parasite culture growth at 24 h (purple bars), 48 h (blue bars), and 72 h (yellow bars) using the IC₁₀₀ of ELQ-316, and at 8 days (green bars). a B. bovis, b B. bigemina, c B. caballi, d T. equi. Upper panel of each figure part represents 0.2% PPE and lower panel represents 2% PPE. “0” represents parasites grown without the addition of ELQ-316. Assays were carried out in triplicate and the error bars indicate standard error deviation. Asterisk (*) represents statistically significant difference between cultures grown without and with ELQ-300 at P < 0.05, using Student’s t-test

Fig. 5

Percentage of cell viability over a period of 72 h after incubation with the IC₅₀ and IC₁₀₀of ELQ-300 and ELQ-316. Cells+Med Peripheral blood mononuclear cells (PBMC) cultivated without the addition of the ELQ compounds. Cells in cultured in DMSO and Draxxin® were used as a negative control; concanavalin A (ConA) was used as a positive control for cell proliferation. a Bovine PBMC, b horse PBMC. Bovine and horse PBMC assays were carried out in triplicate and the error bars indicate standard error deviation. Asterisk (*) represents statistically significant differences compared to PBMC cultivated in medium only at P < 0.05, using Student’s t-test