In vitro quantitative analysis of (3)H-uracil incorporation by Sarcocytis neurona to determine efficacy of anti-protozoal agents.
Parasite-specific incorporation of (3)H-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 1.0microg/ml PYR), sulfadiazine (5microg/ml; SDZ), sulfamethoxazole (5microg/ml; SMZ), diclazuril (100ng/ml; DCZ), atovaquone (0.04ng/ml; ATQ), tetracycline (5microg/ml; TET) and the herbicide glyphosate (1.5 and 4.5mM; GLY) were studied with varying S. neurona parasite densities (2x10(1)-1.2x10(6)merozoites/well). A microtiter plate format was used to test these compounds, and incorporation of (3)H-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 9.0x10(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 (3)H-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.
Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia 65211, USA. marshae@missouri.edu
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Marsh
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Mullins
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Lakritz
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D053626:Atovaquone; D002417:Cattle; D004261:DNA Replication; D003670:Deer; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006540:Herbicides / Q000494:pharmacology; D008297:Male; D009285:Naphthoquinones / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D011739:Pyrimethamine / Q000494:pharmacology; D012098:Reproduction; D012522:Sarcocystis / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D013411:Sulfadiazine / Q000494:pharmacology; D013420:Sulfamethoxazole / Q000494:pharmacology; D013752:Tetracycline / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology; D014316:Tritium; D014498:Uracil / Q000378:metabolism*; D000097797:Glyphosate
D000981:Antiprotozoal Agents; D053626:Atovaquone; D005998:Glycine; D006540:Herbicides; D009285:Naphthoquinones; D009570:Nitriles; D011739:Pyrimethamine; D013411:Sulfadiazine; D013420:Sulfamethoxazole; D013752:Tetracycline; D014227:Triazines; D014316:Tritium; D014498:Uracil; D000097797:Glyphosate; C057884:diclazuril