Examination of the activities of 43 chemotherapeutic agents against Neospora caninum tachyzoites in cultured cells

David S. Lindsay From the Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519 (Lindsay, Rippey, Cole, Parsons, Blagburn), USDA, ARS, LPSI, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705 (Dubey), and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill NC 27599 (Tidwell).

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Natasha S. Rippey From the Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519 (Lindsay, Rippey, Cole, Parsons, Blagburn), USDA, ARS, LPSI, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705 (Dubey), and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill NC 27599 (Tidwell).

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Rebecca A. Cole From the Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519 (Lindsay, Rippey, Cole, Parsons, Blagburn), USDA, ARS, LPSI, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705 (Dubey), and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill NC 27599 (Tidwell).

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Lisa C. Parsons From the Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519 (Lindsay, Rippey, Cole, Parsons, Blagburn), USDA, ARS, LPSI, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705 (Dubey), and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill NC 27599 (Tidwell).

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J. P. Dubey From the Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519 (Lindsay, Rippey, Cole, Parsons, Blagburn), USDA, ARS, LPSI, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705 (Dubey), and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill NC 27599 (Tidwell).

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Richard R. Tidwell From the Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519 (Lindsay, Rippey, Cole, Parsons, Blagburn), USDA, ARS, LPSI, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705 (Dubey), and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill NC 27599 (Tidwell).

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Byron L. Blagburn From the Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL 36849-5519 (Lindsay, Rippey, Cole, Parsons, Blagburn), USDA, ARS, LPSI, Parasite Biology and Epidemiology Laboratory, Beltsville, MD 20705 (Dubey), and Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill NC 27599 (Tidwell).

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Summary

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.

Summary

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.

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