Author ORCID Identifier
Date Available
6-7-2024
Year of Publication
2024
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Agriculture, Food and Environment
Department/School/Program
Veterinary Science
First Advisor
Daniel K. Howe
Abstract
Equine protozoal myeloencephalitis (EPM) is an important neurologic disease of horses in the Americas. It is caused by the apicomplexan parasite Sarcocystis neurona and less commonly by the related parasite Neospora hughesi. Current antiprotozoal treatment relies on the use of either folate inhibitors (sulfadiazine/pyrimethamine) or triazine drugs (diclazuril/ponazuril), with a success rate of 60-70% regardless of the drug of choice. Successful treatment is defined as the improvement of at least one ataxia grade following the modified Mayhew scale, which often is not enough for the horse to return to the previous level of performance. The goals of this dissertation are to i) identify the molecular target of the triazine drugs in apicomplexan parasites, and ii) explore the pharmacokinetics (PK) of a novel antiprotozoal drug class in horses. To address the first goal, a chemical mutagenesis scheme was employed to generate diclazuril-resistant S. neurona clones that were analyzed by whole genome sequencing and nucleotide variant identification. Comparison with the reference genome and wild-type controls revealed a single nucleotide variant on the gene SN3_01000220, which encodes a major facilitator superfamily (MFS) transporter. To confirm this prediction, a CRISPR-based approach was designed to mutate and add an epitope tag to the homologous MFS gene in Toxoplasma gondii, a closely related parasite that is more amenable to genetic manipulation. The mutated T. gondii clone was able to grow at 5 µg/ml of diclazuril, 1000X the effective concentration, thus confirming that the missense mutation in the MFS protein confers resistance. Moreover, immunofluorescence microscopy showed that the MFS transporter was localized to the apicoplast, an organelle unique to apicomplexan parasites that originated from a photosynthetic endosymbiont. Additionally, abnormal apicoplast morphology was observed following diclazuril treatment of S. neurona. Further research is needed to define how diclazuril interacts with this transporter and its biological function. Bumped-kinase inhibitors (BKIs) are a novel class of antiprotozoal drugs that are currently under investigation in a multicenter study for the treatment of parasitic diseases in humans and livestock species. The BKI-1708 half-maximal inhibitory concentration for S. neurona in vitro was determined to be 42 nM, and a preliminary intravenous PK revealed that levels in horse plasma began at 4 µM, with an initial distribution phase followed by a terminal phase, and a half-life of 2 hours. The follow-up single and multi-dose oral PK studies determined that BKI-1708 had a bioavailability estimated to be 50%, with a half-life of 15 hours. Steady-state levels were rapidly achieved with plasma peaks of 1 µM and trough levels of 0.3 µM, which are in excess to inhibit S. neurona replication. No significant side effects were noted based on daily clinical exams and bloodwork. These results demonstrate that the anti-protozoal BKI-1708 is well tolerated by horses and is thus a promising compound for treating EPM. The next steps include the assessment of drug levels within the central nervous system. If effective levels are found, a therapeutic dosing regimen will be developed and horses with an antemortem diagnosis supportive of EPM recruited for a clinical trial with the drug being compared to ponazuril.Future avenues of research on EPM should expand the understanding of the immune response to S. neurona so that immunomodulatory and regenerative approaches can be developed to complement the current antiprotozoal-based therapies.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2024.243
Funding Information
1 - Amerman Family Equine Research Endowment
2 - United States Department of Agriculture - National Institute of Food and Agriculture (USDA-NIFA) AFRI grant # 2020-67015-30881
3 - Merck Animal Health Research Fellowship (2022-2024)
Recommended Citation
de Assis Rocha, Izabela, "Current and New Perspectives on Equine Protozoal Myeloencephalitis Therapeutics" (2024). Theses and Dissertations--Veterinary Science. 65.
https://uknowledge.uky.edu/gluck_etds/65
