title: Demonstration of synergistic effects of sulfonamides and dihydrofolate reductase/thymidylate synthase inhibitors against Neospora caninum tachyzoites in cultured cells, and characterization of mutants resistant to pyrimethamine.
abstract: OBJECTIVE
  To examine the efficacies of combinations of 7 sulfonamides and 5 dihydrofolate reductase/thymidylate synthase (DHFR/TS) inhibitors against tachyzoites of Neospora caninum in cultured cells. Mutant tachyzoites that were resistant to pyrimethamine were produced and examined for resistance to other DHFR/TS inhibitors.
  
  
  METHODS
  After 5 days of treatment, a cell culture flask lesion-based assay was used to determine efficacies of combinations of sulfonamides and DHFR/TS inhibitors against N caninum tachyzoites and to evaluate the sensitivity of pyrimethamine-resistant mutants of N caninum to test agents. Cultured cells that were infected with the appropriate strains of N caninum and treated or not treated (controls) with test agents were examined. Mutations were induced by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine or by selection for growth in permissive concentration of pyrimethamine.
  
  
  RESULTS
  Synergism was detected for combinations of pyrimethamine, ormetoprim, trimethoprim, or diaveridine with the sulfonamides. Methotrexate did not have improved efficacy when combined with sulfonamides. Two mutants were produced that were resistant to pyrimethamine. Both mutants were resistant to other DHFR/TS inhibitors. Both mutants remained resistant to pyrimethamine in the absence of continuous exposure to the agent, indicating that the induced resistance was stable. Synergism was detected for combinations of DHFR/TS inhibitors and sulfonamides against these pyrimethamine-resistant mutants.
  
  
  CONCLUSIONS
  Combinations of suboptimal concentrations of sulfonamides with suboptimal concentrations of DHFR/TS inhibitors results in improved efficacy of the agents in a cell culture assay. Stable resistance to pyrimethamine can be induced in N caninum tachyzoites by use of chemical mutagenesis or by selection.
  
  
  CONCLUSIONS
  In vitro evidence indicated that combination treatment, using sulfonamides and DHFR/TS inhibitors, may be effective in treating neosporosis.
authors.affiliation: Department of Pathobiology, Auburn University, AL 36849-5519, USA.
authors.forename: D S
authors.initials: DS
authors.lastname: Lindsay
authors.forename: J M
authors.initials: JM
authors.lastname: Butler
authors.forename: N S
authors.initials: NS
authors.lastname: Rippey
authors.forename: B L
authors.initials: BL
authors.lastname: Blagburn
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology*; D002460:Cell Line; D003049:Coccidiostats / Q000494:pharmacology*; D004351:Drug Resistance; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006801:Humans; D008297:Male; D018688:Neospora / Q000187:drug effects* / Q000254:growth & development; D011739:Pyrimethamine / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012867:Skin; D013449:Sulfonamides / Q000494:pharmacology*; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors; D014295:Trimethoprim / Q000494:pharmacology
chemical_list: D000890:Anti-Infective Agents; D003049:Coccidiostats; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D011743:Pyrimidines; D013449:Sulfonamides; C033222:diaveridine; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; C100125:ormetoprim; D011739:Pyrimethamine