In vitro quantitative analysis of 3H-uracil incorporation by Sarcocytis neurona to determine efficacy of anti-protozoal agents

Abstract

Parasite-specific incorporation of 3H-uracil was used to assess the replication of Sarcocystis neurona, a protozoal parasite associated with equine protozoal myeloencephalitis (EPM). Anti-protozoal drugs, pyrimethamine (0.01, 0.1 and 1.0 μg/ml PYR), sulfadiazine (5 μg/ml; SDZ), sulfamethoxazole (5 μg/ml; SMZ), diclazuril (100 ng/ml; DCZ), atovaquone (0.04 ng/ml; ATQ), tetracycline (5 μg/ml; TET) and the herbicide glyphosate (1.5 and 4.5 mM; GLY) were studied with varying S. neurona parasite densities ( 2×10 1–1.2×10 6 merozoites/ well ). A microtiter plate format was used to test these compounds, and incorporation of 3H-uracil was determined using a semi-automated plate harvester and liquid scintillation counter. When PYR, DCZ, ATQ, SMZ, SDZ, and TET were tested, the assay was most reliable when parasite densities were greater than 9.0×10 4 individual merozoites per well. When the herbicide GLY was tested, as few as 900 individual merozoites were sufficient to demonstrate reduction in parasite proliferation. Of the anti-protozoal drugs commonly used to treat EPM, PYR was the most potent anti- S. neurona agent tested. The herbicide GLY appears to be more potent than all of the other compounds tested in vitro; however information regarding in vivo use of GLY is not available, and central nervous system penetration by this compound is unlikely. Incorporation of 3H-uracil by replicating S. neurona is quantitative and can be used in a semi-automated assay. This in vitro assay is capable of high throughput screening of candidate drugs that may have applications in a clinical setting. Further studies using a wider range of drug concentrations with optimal numbers of merozoites are necessary to determine true potency of these agents.