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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.

Design and Procedures

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-nitrosoguanidme or by selection for growth in permissive concentration of pyrimethamine.


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.


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.

Clinical Relevance

In vitro evidence indicated that combination treatment, using sulfonamides and DHFR/TS inhibitors, may be effective in treating neosporosis. (Am J Vet Res 1996;57:68-72)

Free access
in American Journal of Veterinary Research


Neospora caninum causes serious disease in dogs, and it, or a similar parasite, is a major cause of abortion in cattle. Little is known about the susceptibility of this protozoan to antimicrobial agents. We studied several antimicrobial agents to determine which classes might have activity against this parasite. We also determined whether activity of such agents was coccidiocidal or coccidiostatic. A 2-day of treatment, monoclonal antibody-based enzyme immunoassay and a 5-day of treatment, cell culture flask (ccf), lesion-based assay were developed to examine the ability of test agents to inhibit tachyzoite multiplication. Seven sulfonamides were examined, with the following activities observed: sulfathiazole ≥ sulfamethoxazole > sulfadiazine > sulfaquinoxaline ≥ sulfamethazine > sulfadimethoxine > sulfamerazine. Dapsone, a sulfone, had little activity. Six dihydrofolate reductase/thymidylate synthase inhibitors were examined, with the following activities observed: piritrexim > pyrimethamine > ormetoprim > trimethoprim = diaveridine > methotrexate. Six ionophorous antibiotics were examined; lasalocid, maduramicin, monensin, narasin, and salinomycin had equivalent activities, but alborixin was toxic for host cells at the lowest concentration examined. Three macrolide antibiotics—azithromycin, clarithromycin, and erythromycin—were examined and had equivalent activities. Two tetracycline antibiotics, doxycycline and minocycline, were examined and had equivalent activities. Three lincosamide antibiotics were examined, with the following activities observed: clindamycin hydrochloride > clindamycin phosphate > lincomycin hydrochloride. Pentamidine and 6 of its analogs were examined, and only hexamidine and 1,4-Di[4-(2-imidazolinyl)-2-methoxy-phenoxy]butane had activity. Eight miscellaneous antiprotozoal agents were examined for activity. Amprolium, metronidazole, paromomycin, and roxarsone had little activity. Arprinocid, diclazuril, nitrofurazone, and robenidine had good activity. Eleven agents were examined in both assays, whereas 32 agents were examined in the ccf assay only. The enzyme immunoassay and ccf assay provided similar results for agents that rapidly killed tachyzoites. However, agents that inhibited development, but were not rapidly fatal for tachyzoites, had better activity in the ccf assay. Of the classes of agents examined, the dihydrofolate reductase/thymidylate synthase inhibitors, 2 of the 6 pentamidine analogs, and the ionophores were coccidiocidal and the sulfonamides, macrolides, tetracyclines, and lincosamides were coccidiostatic. Of the miscellaneous agents examined, arprinocid, nitrofurazone, and robenidine were coccidiocidal and diclazuril was coccidiostatic.

Free access
in American Journal of Veterinary Research