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

Parasite-specific incorporation of H-3-uracil was used to assess the replic ation of Sarcocystis neurona, a protozoal parasite associated with equine p rotozoal myeloencephalitis (EPM). Anti-protozoal drugs, pyrimethamine (0.01 , 0.1 and 1.0 mug/ml PYR), sulfadiazine (5 mug/ml; SDZ), sulfamethoxazole ( 5 mug/ml; SMZ), diclazuril (100 ng/ml; DCZ), atovaquone (0.04 ng/ml; ATQ), tetracycline (5 mug/ml; TET) and the herbicide glyphosate (1.5 and 4.5 mM; GLY) were studied with varying S. neurona parasite densities (2 x 10(1)-1.2 x 10(6) merozoites/well), A microtiter plate format was used to test these compounds, and incorporation of H-3-uracil was determined using a semi-aut omated 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 x 10(4) individual merozoites per well. Whe n the herbicide GLY was tested, as few as 900 individual merozoites were su fficient to demonstrate reduction in parasite proliferation. Of the anti-pr otozoal drugs commonly used to treat EPM, PYR was the most potent anti-S. n eurona agent tested. The herbicide GLY appears to be more potent than all o f the other compounds tested in vitro; however information regarding in viv o use of GLY is not available, and central nervous system penetration by th is compound is unlikely, Incorporation of H-3-uracil by replicating S, neur ona is quantitative and can be used in a semi-automated assay. This in vitr o assay is capable of high throughput screening of candidate drugs that may have applications in a clinical setting. Further studies using a wider ran ge of drug concentrations with optimal numbers of merozoites are necessary to determine true potency of these agents. (C) 2001 Elsevier Science B.V. A ll rights reserved.