Activities of Endochin-Like Quinolones Against in vitro Cultured Besnoitia besnoiti Tachyzoites.

Naja Eberhard,Samuel Francisco,Joachim Müller,J Stone Doggett,Andrew Hemphill,Aaron Nilsen,Mike Riscoe,Alexandre Leitao,Rolf Winter,Vreni Balmer,Soviti Pou,Norbert Müller

doi:10.3389/fvets.2020.00096

Abstract

Endochin-like quinolones (ELQs) potently inhibit the proliferation of Plasmodium, Toxoplasma, Neospora, and Babesia by targeting the cytochrome b Qo and Qi sites and interfering with oxidative phosphorylation and pyrimidine biosynthesis. The activities of 14 different ELQs were assessed against B. besnoiti tachyzoites grown in human foreskin fibroblasts (HFF) by quantitative real time PCR. The values for 50% proliferation inhibition (IC50) of five ELQs were determined in a 3-days growth assay after an initial screen of 12 ELQs at 0.01, 0.1, and 1 μM. The IC50s of ELQ-121, -136, and -316 were 0.49, 2.36, and 7.97 nM, respectively. The IC50s of ELQs tested against B. besnoiti were higher than IC50s previously observed for P. falciparum and T. gondii. However, the B. besnoiti cytochrome b sequence and the predicted Qo and Qi ELQ binding sites in the Toxoplasma, Neospora, and Besnoitia cytochrome b are virtually identical, suggesting that the differences in ELQ susceptibility are not due to variations in the substrate binding sites. TEM of ELQ-treated parasites primarily demonstrated alterations within the parasite mitochondrion, profound thickening of the nuclear membrane, as well as increased vacuolization within the tachyzoite cytoplasm. Long-term treatment assays of intracellular B. besnoiti with ELQs for up to 20 days followed by the release of drug pressure caused a substantial delay in parasite growth and proliferation while ELQs were present, but parasite proliferation resumed days after ELQs were removed. Interestingly, structural alterations persisted after ELQ removal and parasite proliferation was slowed. These findings provide a basis for further in vivo studies of ELQs as therapeutic options against B. besnoiti infection.

Highlights

Besnoitia besnoiti is a cyst-forming coccidian parasite (1), which is closely related to Neospora caninum and Toxoplasma gondii and is the causative agent of bovine besnoitiosis (2, 3)
For a preliminary efficacy assessment, B. besnoiti infected human foreskin fibroblasts (HFF) were exposed to the 14 Endochin-like quinolones (ELQs) at 1, 0.1, and 0.01 μM
ELQ-400 has been shown to exhibit outstanding in vivo activity, better than atovaquone, in an acute toxoplasmosis mouse model (22), and there is evidence that it acts as a dual Qo and Qi site inhibitor (21)

Summary

Introduction

Besnoitia besnoiti is a cyst-forming coccidian parasite (1), which is closely related to Neospora caninum and Toxoplasma gondii and is the causative agent of bovine besnoitiosis (2, 3). Bovine besnoitiosis was originally restricted to endemic areas in Portugal, Spain, and South of France (4). ELQs and Besnoitia besnoiti expansion and increased number of cases, the European Food Safety Authority (EFSA) has proclaimed besnoitiosis as a reemerging disease (6, 7). Similar to other cyst-forming coccidian parasites, B. besnoiti is suspected to have an indirect life cycle in which a carnivore represents the definitive host and cattle as well as wild bovids serve as intermediate hosts. Cats have been shown to be the definitive host of several other Besnoitia species (B. darlingi, B. wallacei, B. orictofelisi, and B. neotomofelis), the definitive host of B. besnoiti has not been identified (1)

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