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.

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. 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-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. CLINICAL RELEVANCE: In vitro evidence indicated that combination treatment, using sulfonamides and DHFR/TS inhibitors, may be effective in treating neosporosis.

Abortions, fetal death, and stillbirths in pregnant pygmy goats inoculated with tachyzoites of Neospora caninum.

Neospora caninum-induced abortion is a major production problem in the dairy cattle industry in the United States and worldwide. Abortions attributable to naturally acquired N caninum infection also have been observed in pygmy goats. We studied experimentally induced infections with N caninum in pregnant pygmy does to determine whether abortions attributable to N caninum infection would occur after inoculation. Seven pregnant pygmy does (1 control doe and 6 inoculated with N caninum) were studied. The control doe remained clinically normal throughout the study and delivered 2 healthy kids. Abortion, fetal death, and stillbirths were observed in some pregnant does inoculated with N caninum. Two pregnant pygmy does inoculated with N caninum early in gestation (day 51) had fetuses that died and were aborted, or died and were reabsorbed. Neospora caninum tachyzoites and lesions were observed in the brain, spinal cord, and heart of aborted fetuses; parasites also were isolated from the placenta. Four additional pregnant pygmy does (2 inoculated at mid-gestation [day 85], and 2 at late gestation [day 127]) did not abort after inoculation. However, 1 doe inoculated during mid-gestation delivered a stillborn fetus that had died about 1 week prior to parturition. This kid was congenitally infected with N caninum. Neospora caninum was isolated from the placentas of all inoculated does examined. Neonatal neosporosis was not observed in live-born kids, nor were stages of N caninum isolated from any live-born kid. Does did not undergo abortion or have congenitally infected kids when they were rebred and evaluated for neosporosis.

Ultrastructural effects of diclazuril against Toxoplasma gondii and investigation of a diclazuril-resistant mutant.

Diclazuril is an anticoccidial that inhibits tachyzoite production of the RH strain of Toxoplasma gondii by > 97% at a concentration of 0.005 micrograms/ml. The effects of 1.0 microgram/ml diclazuril on the development of 3 strains (RH and 2 tissue cyst formers, GT-1, and WTD-3) of T. gondii in vitro was examined using transmission electron microscopy. The effects of diclazuril were not noted until 2 days after treatment. Treatment with diclazuril interfered with endodyogeny and resulted in the production of multinucleate (> 2 nuclei) meront stages. Up to 12 nuclei were observed in some meronts. Tachyzoites attempted to bud from the surfaces of these stages but division was apparently blocked by the action of diclazuril. The parasitophorous vacuole (PV) enclosing developing stages became hypertrophic. As the meront stages degenerated the PV became filled with membranous material and the cytoplasmic contents of lysed stages. Formation of tissue cysts by the GT-1 and WTD-3 strains of T. gondii was not prevented by treatment with diclazuril. A mutant of the RH strain that was resistant to 1.0 microgram/ml diclazuril was selected by progressive culture in permissive levels of the agent. This DicR-1 mutant produced significantly fewer tachyzoites (P < 0.05) in vitro than its parent strain. Resistance to diclazuril appears to be stable because reversion to sensitivity to the agent did not occur after 50 passages in the absence of drug pressure. The DicR-1 mutant was somewhat less pathogenic for mice (70% mortality) than its parent RH strain (100% mortality) and persisted as tissue cysts in the brains of mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of acute Toxoplasma gondii infections in mice with diclazuril or a combination of diclazuril and pyrimethamine.

Diclazuril is a benzeneacetonitrile anticoccidial that is also effective in the prevention of toxoplasmosis. The present study was conducted to examine the efficacy of diclazuril alone or in combination with pyrimethamine for the treatment of acute toxoplasmosis in mice. Diclazuril administered at 10 mg/kg beginning 6 days after inoculation and given for 10 days protected 90% of mice over a 56-day observation period. Treatment with diclazuril at 1.0 or 0.5 mg/kg protected 20 and 0% of mice, respectively. Treatment with pyrimethamine at 12.5 or 6.0 mg/kg protected 60 and 0% of mice, respectively. When diclazuril (1.0 or 0.5 mg/kg) was combined with pyrimethamine (12.5 mg/kg) all mice survived the 56-day observation period. When diclazuril (1.0 or 0.5 mg/kg) was combined with pyrimethamine (6.0 mg/kg) 30 and 10% of mice, respectively, survived the 56-day observation period.

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

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 > or = sulfamethoxazole > sulfadiazine > sulfaquinoxaline > or = 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.(ABSTRACT TRUNCATED AT 250 WORDS)